INTRATHECAL ADMINISTRATION OF ADENO-ASSOCIATED-VIRAL VECTORS FOR GENE THERAPY

Abstract

A composition comprising at least one AAV vector formulated for intrathecal delivery to the central nervous system is described. The composition comprises at least one expression cassette which contains sequences encoding an immunoglobulin construct linked to expression control sequences therefor and a pharmaceutically acceptable carrier. The immunoglobulin construct may be an immunoglobulin modified to have decreased or no measurable affinity for neonatal Fc receptor (FcRn).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Nonprovisional patent application Ser. No. 15/771,481, filed Apr. 27, 2018, which is a 371 of International Patent Application No. PCT/US2016/058968, filed Oct. 27, 2016, now expired, and which claims benefit of the priority of U.S. Provisional Patent Application No. 62/247,498, filed Oct. 28, 2015, now expired, which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] Biomedical and pharmaceutical researchers have worked to devise new and more effective therapeutics to treat diseases affecting the central nervous system. However, the biology of the central nervous system itself, including the effectiveness of the blood-brain barrier in protecting the brain, poses a profound challenge to drug delivery. This leads to a lack of available treatment for many central nervous system diseases, such as stroke, neurodegenerative disorders, and brain tumors.

[0003] Neurodegeneration is the umbrella term for the progressive loss of structure or function of neurons, including death of neurons. Many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's, Alzheimer's, and Huntington's occur as a result of neurodegenerative processes. A variety of therapies have been described for treatment of such neurodegenerative diseases, including monoclonal antibody therapy.

[0004] Adeno-associated virus (AAV) is a replication-deficient parvovirus, the single-stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs). The nucleotide sequence of the AAV serotype 2 (AAV2) genome is presented in Srivastava et al., J Virol, 45: 555-564 (1983) as corrected by Ruffing E T ah, J Gen Virol, 75: 3385-3392 (1994). Cis-acting sequences directing viral DNA replication (rep), encapsulation/packaging and host cell chromosome integration are contained within the ITRs. Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes. The two rep promoters (p5 and pi 9), coupled with the differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene. Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome. The cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins. A single consensus polyadenylation site is located at map position 95 of the AAV genome. The life cycle and genetics of AAV are reviewed in Muzyczka. Current Topics in Microbiology and Immunology, 158: 97-129 (1992). The coding sequences of the AAV genome can be provided in trans, making it possible to generate AAV vectors carrying a gene of interest rather than the endogenous viral genes. These AAV vectors are capable of gene transfer in vivo. Multiple serotypes of AAV exist and offer varied tissue tropism. Known serotypes include, for example, AAV1, AAV2, AAV3. AAV4, AAV5, AAV6, AAV7, AAV8. AAV9, AAV10 and AAV1, AAV9 is described in U.S. Pat. No. 7,198,951 and in Gao et al, J. Virol, 78: 6381-6388 (2004).

[0005] Intrathecal administration of an AAV vector carrying a single chain secretable ScFv antibody to target SOD1 has been described as a potential therapeutic approach for ALS. See, e.g., P Patel et al, Molecular Therapy (2014): 22, 3, 498-510.

[0006] What are needed are other methods for delivering immunoglobulin constructs, including without limitation, full-length antibodies, to the central nervous system.

SUMMARY OF THE INVENTION

[0007] A composition is provided which comprises an AAV vector formulated for intrathecal delivery to the central nervous system, wherein said composition comprises at least one expression cassette which contains sequences encoding an immunoglobulin product for delivery to the CNS operably linked to expression control sequences therefor and a pharmaceutically acceptable carrier and/or excipient. In one aspect, the composition is administered in the absence of chemical or physical disruption of the blood brain barrier. In one example, the immunoglobulin construct comprises an immunoglobulin modified to have decreased or no measurable affinity for neonatal Fc receptor (FcRn).

[0008] In one aspect, a composition is described which comprises an AAV vector formulated for intrathecal delivery for treatment of a condition of the central nervous system, wherein said composition comprises an AAV vector comprising an AAV capsid which targets a cell of the central nervous system, the capsid having packaged therein at least one AAV inverted terminal repeat sequence and sequences encoding an immunoglobulin construct operably linked to expression control sequences therefor, the composition further comprising a pharmaceutically acceptable carrier and/or excipient. Suitably, the encoded immunoglobulin is mutated or engineered to have decreased or no measurable affinity for neonatal Fc receptor (FcRn).

[0009] Use of a composition comprising at least one AAV vector stock encoding the immunoglobulin construct useful in treatment of a neurological disorder, and/or an infectious disease of the central nervous system, is provided herein.

[0010] In another aspect, a method for treatment of Alzheimer's Disease is provided which involves intrathecal delivery of an AAV vector composition as described herein, in which at least one AAV vector stock expresses an immunoglobulin specific for a A.beta., beta secretase, and/or the tau protein, to subject in need thereof.

[0011] In yet another aspect, a method for treatment of ALS is provided which involves intrathecal delivery of an AAV vector composition as described herein, in which at least one AAV vector stock expresses an immunoglobulin specific for ALS enzyme superoxide dismutase 1 (SOD1) and variants thereof, provided the anti-SOD1 antibody is other than an ScFv fragment, a Derlin-1-binding region, and/or an antibody construct against neurite outgrowth inhibitor.

[0012] In still another embodiment, a method for treatment of Parkinson's Disease or related synucleinopathies is provide which involves intrathecal delivery of an AAV vector composition as provided herein, in which at least one AAV vector stock encodes one or more leucine-rich repeat kinase 2 antibody, dardarin (LRRK2) antibody, alpha-synuclein antibody, and/or DJ-1 (PARK7) antibody.

[0013] In yet a further aspect, a method for treatment of multiple sclerosis is provided herein which involves intrathecal delivery of an AAV vector composition as provided herein, in which composition at least one vector stock encodes an immunoglobulin directed against one or more of an a4-integrin, CD20, CD25, IL12, p40+IL23p40, LINGO-1, CD40, and rHIgM22, CD52, IL17, CD19, and/or SEMA4D.

[0014] In another aspect, a method for treatment of infectious disease of the central nervous system is provided which involves intrathecal delivery of an AAV vector composition as provided herein, in which composition at least one vector stock encodes an immunoglobulin directed against the pathogen which causes said infectious disease. Examples, without limitation, include one or more immunoglobulins directed against one or more of Mycobacterium tuberculosis (tuberculosis), Neisseria meningitides (meningitis), Streptococcus pneumonia, Listeria monocytogens (listeriosis), Borrelia burdorferia (lyme disease), human deficiency virus (acquired immunodeficiency syndrome), a herpes family viruses, varicella zoster virus, Epstein-Barr virus (EBV), cytomegalovirus, and/or JC virus. Other examples of immunoglobulin targets are provided elsewhere in this application and are incorporated by reference herein.

[0015] In one aspect a method is provided for treatment of prion related diseases which comprises intrathecal delivery of an AAV vector composition, in which at least one vector stock encodes an immunoglobulin directed one or more of major prion protein, or PrPSc.

[0016] Still other aspects and advantages of the invention will be readily apparent from the following detailed description of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1A is a graph which shows concentration of a rhesus macaque derived immunoadhesin (201IA) in cerebrospinal fluid (CSF) for two animals receiving an AAV9 vector expressing the immunoadhesin under a chicken .beta.-actin (CB) promoter delivered intrathecally by suboccipital puncture. CSF was collected periodically following vector administration, with 400 days being the last collection time point shown. The scale for CSF is up to 800 ng/mL.

[0018] FIG. 1B is a graph which shows concentration of a rhesus macaque derived immunoadhesin (201IA) in serum for two animals receiving an AAV9 vector expressing the immunoadhesin under a chicken .beta.-actin (CB) promoter delivered intrathecally by suboccipital puncture. Serum was collected periodically following vector administration, with 400 days being the last collection time point shown. The concentration scale for serum is up to 120 .mu.g/mL.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The compositions and regimens described herein are useful for delivery of immunoglobulin constructs to the central nervous system. Compositions described herein comprising AAV having immunoglobulin constructs for intrathecal delivery to the central nervous system (CNS).

[0020] As used herein, an immunoglobulin construct (including antibody or antibody fragment as defined herein) encodes a polypeptide-based moiety which binds to a cell-surface antigen or receptor located within the central nervous system. Such a receptor may be located on a bacteria, virus, fungus, or other pathogen which has infected the central nervous system, and/or proteins associated with a disorder of the central nervous system and/or such a pathogen, e.g., secreted proteins and/or protein aggregates.

[0021] The term "immunoglobulin" is used herein to include antibodies, functional fragments thereof, and immunoadhesins. Antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, camelid single domain antibodies, intracellular antibodies ("intrabodies"), recombinant antibodies, multispecific antibody, antibody fragments, such as, Fv, Fab, F(ab).sub.2, F(ab).sub.3, Fab', Fab'-SH, F(ab').sub.2, single chain variable fragment antibodies (scFv), tandem/bis-scFv, Fc, pFc', scFvFc (or scFv-Fc), disulfide Fv (dsfv), bispecific antibodies (bc-scFv) such as BiTE antibodies; camelid antibodies, resurfaced antibodies, humanized antibodies, fully human antibodies, single-domain antibody (sdAb, also known as NANOBODY.RTM.), chimeric antibodies, chimeric antibodies comprising at least one human constant region, and the like. "Antibody fragment" refers to at least a portion of the variable region of the immunoglobulin that binds to its target, e.g., the cell-surface antigen or receptor.

[0022] In one embodiment, a composition as described herein provides for AAV-mediated delivery of an immunoglobulin which includes a fragment crystallizable region (Fc portion), including, such as is present in a full-length antibody, a bispecific antibody, an immunoadhesin [containing the immunoglobulin constant domains, and typically also the hinge and Fc regions], a monoclonal antibody, a heavy chain camelid or shark immunoglobulins Use of AAV to deliver full-length antibodies and two antibody combinations have been described, e.g., in "Compositions Comprising AAV Expressing Dual Antibody Constructs and Uses Thereof", International Application No. PCT/US15/30533, filed May 13, 2015, "Methods and Compositions for Treating Metastatic Breast Cancer and Other Cancers in the Brain", International Application No. PCT/US15/27491, filed Apr. 24, 2015, and which are incorporated by reference herein. Optionally, a composition may contain two or more different AAV-immunoglobulin constructs as described herein.

[0023] In another embodiment, a composition as described herein provides for AAV-mediated delivery of an immunoglobulin which excludes an Fc portion, e.g., a Fab (fragment antigen-binding fragment, typically formed by papain digestion of Ab), a F(ab').sub.2 fragment (containing two antigen binding fragments, typically formed by pepsin digestion of Ab), a Fab'fragment (typically formed by reduction of F(ab').sub.2), Fab'-SH, a F(ab).sub.3 (a trispecific antibody fragment), an Fv (immunoglobulin containing only the two variable domains), a single domain antibody (sdAb or V.sub.HH nanobody), e.g., a camelid or shark antibody, or an scFv construct. Such a composition may include two or more different AAVscFv constructs.

[0024] The term "heterologous" when used with reference to a protein or a nucleic acid indicates that the protein or the nucleic acid comprises two or more sequences or subsequences which are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid. For example, in one embodiment, the nucleic acid has a promoter from one gene arranged to direct the expression of a coding sequence from a different gene. Thus, with reference to the coding sequence, the promoter is heterologous.

[0025] As used herein, an "expression cassette" refers to a nucleic acid molecule which comprises an immunoglobulin gene(s) (e.g., an immunoglobulin variable region, an immunoglobulin constant region, a full-length light chain, a full-length heavy chain or another fragment of an immunoglobulin construct), promoter, and may include other regulatory sequences therefor, which cassette may be delivered via a genetic element (e.g., a plasmid) to a packaging host cell and packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such an expression cassette for generating a viral vector contains the immunoglobulin sequences described herein flanked by packaging signals of the viral genome and other expression control sequences such as those described herein.

[0026] As used herein, a "vector stock" or an "AAV vector stock" refers to population of genome copies of an AAV viral particle having packaged therein a non-AAV sequence which encodes an immunoglobulin(s) as defined herein. Suitably, a vector stock includes a sufficient number of genome copies (GC) of the recombinant AAV vector to achieve a desired physiologic effect. Where a desired physiologic effect is achieved, the amount of vector stock in a composition, dose, or regimen, may be referred to as an "effective amount" of rAAV vector or vector stock.

[0027] Unless otherwise specified, the "central nervous system" refers to the spinal cord and brain and contrasts with the "peripheral nervous system" which excludes the spinal cord and brain. There are different cell types within the central nervous system, including neuronal cells and glial cells. The glia in mature systems include astrocytes, oligodendrocytes, and microglial cells. The AAV capsids for the vectors used in the present invention are preferably selected from among those which will transduce and/or express in at least one of these cell types of the central nervous system.

[0028] As used herein, the terms "intrathecal delivery" or "intrathecal administration" refer to a route of administration for drugs via an injection into the spinal canal, more specifically into the subarachnoid space so that it reaches the cerebrospinal fluid (CSF). Intrathecal delivery may include lumbar puncture, intraventricular (including intracerebroventricular (ICV)), suboccipital/intracisternal, and/or C1-2 puncture. For example, material may be introduced for diffusion throughout the subarachnoid space by means of lumbar puncture. In another example, injection may be into the cisterna magna.

[0029] As used herein, the terms "intracisternal delivery" or "intracisternal administration" refer to a route of administration for drugs directly into the cerebrospinal fluid of the cisterna magna cerebellomedularis, more specifically via a suboccipital puncture or by direct injection into the cisterna magna or via permanently positioned tube.

[0030] As described above, the term "about" when used to modify a numerical value means a variation of 10%, unless otherwise specified.

[0031] As used throughout this specification and the claims, the terms "comprise" and "contain" and its variants including, "comprises", "comprising", "contains" and "containing", among other variants, is inclusive of other components, elements, integers, steps and the like. The term "consists of" or "consisting of" are exclusive of other components, elements, integers, steps and the like.

[0032] For expression from an AAV vector, nucleic acid constructs which encode immunoglobulins useful in treatment of one or more neurodegenerative disorders may be engineered or selected for delivery via an AAV composition of the invention. Such disorders may include, without limitation, transmissible spongiform encephalopathies (e.g., Creutzfeld-Jacob disease), Parkinson's disease, amyotropic lateral sclerosis (ALS), multiple sclerosis, Alzheimer's Disease, Huntington disease, Canavan's disease, traumatic brain injury, spinal cord injury (ATI335, anti-nogol by Novartis), migraine (ALD403 by Alder Biopharmaceuticals; LY2951742 by Eli; RN307 by Labrys Biologics), lysosomal storage diseases, stroke, and infectious disease affecting the central nervous system.

[0033] Still other nucleic acids may encode an immunoglobulin which is directed to leucine rich repeat and immunoglobulin-like domain-containing protein 1 (LINGO-1), which is a functional component of the Nogo receptor and which is associated with essential tremors in patients which multiple sclerosis, Parkinson's Disease or essential tremor. One such commercially available antibody is ocrelizumab (Biogen, BIIB033). See. e.g., U.S. Pat. No. 8,425,910.

[0034] In one embodiment, the nucleic acid constructs encode immunoglobulin constructs useful for patients with ALS. Examples of suitable antibodies include antibodies against the ALS enzyme superoxide dismutase 1 (SOD1) and variants thereof (e.g., ALS variant G93A, C4F6 SOD1 antibody); MS785, which directed to Derlin-1-binding region); antibodies against neurite outgrowth inhibitor (NOGO-A or Reticulon 4), e.g., GSK1223249, ozanezumab (humanized, GSK, also described as useful for multiple sclerosis).

[0035] Nucleic acid sequences may be designed or selected which encode immunoglobulins useful in patients having Alzheimer's Disease. Such antibody constructs include, e.g., adumanucab (Biogen), Bapineuzumab (Elan; a humanised mAb directed at the amino terminus of A.beta.); Solanezumab Eli Lilly, a humanized mAb against the central part of soluble A.beta.); Gantenerumab (Chugai and Hoffmann-La Roche, is a full human mAb directed against both the amino terminus and central portions of A.beta.); Crenezumab (Genentech, a humanized mAb that acts on monomeric and conformational epitopes, including oligomeric and protofibrillar forms of A.beta.; BAN2401 (Esai Co., Ltd, a humanized immunoglobulin G1 (IgG1) mAb that selectively binds to A.beta. protofibrils and is thought to either enhance clearance of A.beta. protofibrils and/or to neutralize their toxic effects on neurons in the brain); GSK 933776 (a humanised IgG1 monoclonal antibody directed against the amino terminus of A.beta.); AAB-001, AAB-002, AAB-003 (Fc-engineered bapineuzumab); SAR228810 (a humanized mAb directed against protofibrils and low molecular weight A.beta.); BIIB037/BART (a full human IgG1 against insoluble fibrillar human A.beta., Biogen Idec), an anti-A.beta. antibody such m266, tg2576 (relative specificity for A.beta. oligomers) [Brody and Holtzman, Annu Rev Neurosci, 2008; 31: 175-193]. Other antibodies may be targeted to beta-amyloid proteins, A.beta., beta secretase and/or the tau protein.

[0036] Illustrated in the examples herein are three anti-A.beta. scFv constructs designed to eliminate the Fc region of full-length anti-amyloid beta (A.beta.). These constructs were designed to reduce the risk of amyloid related imaging abnormalities and/or to limit the exposure of vessels to high monoclonal antibody concentrations. In one embodiment, an illustrative scFV binds fibrillar .beta.-amyloid. See, e.g., the scFV having the amino acid sequence of SEQ ID NO: 8, in which the heavy chain variable region has the sequence of amino acids 21 to 143 and the light chain variable region has the sequence of amino acids 159 to 265. Also encompassed are variants of this scFV. For example, with reference to SEQ ID NO: 8, a different signal sequence (amino acids 1-20), a different linker may be substituted for the Gly-Ser linker (amino acids 144 to 158), or the His tag may be removed (amino acids 266 to 271). Provided herein are nucleic acid sequences encoding the scFV amino acid sequences described herein. See. e.g., SEQ ID NO: 7.

[0037] In another embodiment, an illustrative scFV is directed against the oligomeric, soluble, and fibrillary .beta.-amyloid. See, e.g., the amino acid sequence of SEQ ID NO: 10, having the heavy chain variable region of aa 21 to 131 and the light chain variable region of aa 147 to 258. Also encompassed are variants of this scFV. For example, with reference to SEQ ID NO: 10, a different signal sequence (amino acids 1-20), a different linker may be substituted for the Gly-Ser linker (amino acids 132 to 146), or the His tag may be removed (amino acids 259-264). Provided herein are nucleic acid sequences encoding the scFV amino acid sequences described herein. See, e.g., SEQ ID NO: 9.

[0038] In still another embodiment, an illustrative scFV is directed against soluble .beta.-amyloid. See, e.g., the amino acid sequence of SEQ ID NO: 12, having the heavy chain variable region of aa 21 to 131 and the light chain variable region of aa 147 to 258. Also encompassed are variants of this scFV. For example, with reference to SEQ ID NO: 12, a different signal sequence (amino acids 1-20), a different linker may be substituted for the Gly-Ser linker (amino acids 132 to 146), or the His tag may be removed (amino acids 259-264). Provided herein are nucleic acid sequences encoding the scFV amino acid sequences described herein. See, e.g., SEQ ID NO: 11.

[0039] In still other embodiments, an anti-.beta.-amyloid antibody is derived from an IgG4 monoclonal antibodies to target .beta.-amyloid in order to minimize effector functions, or construct other than an scFv which lacks an Fc region is selected in order to avoid amyloid related imaging abnormality (ARIA) and inflammatory response. In certain of these embodiments, the heavy chain variable region and/or the light chain variable region of one or more of the scFv constructs is used in another suitable immunoglobulin construct as provided herein. These scFV and other engineered immunoglobulins may reduce the half-life of the immunoglobulin in the serum, as compared to immunoglobulins containing Fc regions. Reducing the serum concentration of anti-amyloid molecules may further reduce the risk of ARIA, as extremely high levels of anti-amyloid antibodies in serum may destabilize cerebral vessels with a high burden of amyloid plaques, causing vascular permeability.

[0040] Nucleic acids encoding other immunoglobulin constructs for treatment of patients with Parkinson's disease may be engineered or designed to express constructs, including, e.g., leucine-rich repeat kinase 2, dardarin (LRRK2) antibodies, anti-synuclein and alpha-synuclein antibodies and DJ-1 (PARK7) antibodies. Other antibodies may include, PRX002 (Prothena and Roche) Parkinson's disease and related synucleinopathies. These antibodies, particularly anti-synuclein antibodies may also be useful in treatment of one or more lysosomal storage disease.

[0041] One may engineer or select nucleic acid constructs encoding an immunoglobulin construct for treating multiple sclerosis. Such immunoglobulins may include or be derived from antibodies such as natalizumab (a humanized anti-a4-ingrin, iNATA, Tysabri, Biogen Idec and Elan Pharmaceuticals), which was approved in 2006, alemtuzumab (Campath-1H, a humanized anti-CD52), rituximab (rituzin, a chimeric anti-CD20), daclizumab (Zenepax, a humanized anti-CD25), ocrelizumab (humanized, anti-CD20, Roche), ustekinumab (CNTO-1275, a human anti-IL 12 p40+IL23p40); anti-LINGO-1, and ch5D12 (a chimeric anti-CD40), and rHIgM22 (a remyelinated monoclonal antibody; Acorda and the Mayo Foundation for Medical Education and Research). Still other anti-a4-integrin antibodies, anti-CD20 antibodies, anti-CD52 antibodies, anti-IL17, anti-CD19, anti-SEMA4D, and anti-CD40 antibodies may be delivered via the AAV vectors as described herein.

[0042] AAV-mediated delivery of antibodies against various infections of the central nervous system is also contemplated by the present invention. Such infectious diseases may include fungal diseases such as cryptoccocal meningitis, brain abscess, spinal epidural infection caused by, e.g., Cryptococcus neoformans, Coccidioides immitis, order Mucorales, Aspergillus spp, and Candida spp; protozoal, such as toxoplasmosis, malaria, and primary amoebic meningoencepthalitis, caused by agents such as, e.g., Toxoplasma gondii, Taenia solium, Plasmodium falciparus, Spirometra mansonoides (sparaganoisis), Echinococcus spp (causing neuro hydatosis), and cerebral amoebiasis; bacterial, such as, e.g., tuberculosis, leprosy, neurosyphilis, bacterial meningitis, lyme disease (Borrelia burgdorferi), Rocky Mountain spotted fever (Rickettsia rickettsia), CNS nocardiosis (Nocardia spp), CNS tuberculosis (Mycobacterium tuberculosis), CNS listeriosis (Listeria monocytogenes), brain abscess, and neuroborreliosis; viral infections, such as, e.g., viral meningitis, Eastern equine encephalitis (EEE), St Louis encepthalitis, West Nile virus and/or encephalitis, rabies, California encephalitis virus, La Crosse encepthalitis, measles encephalitis, poliomyelitis, which may be caused by, e.g., herpes family viruses (HSV), HSV-1, HSV-2 (neonatal herpes simplex encephalitis), varicella zoster virus (VZV), Bickerstaff encephalitis, Epstein-Barr virus (EBV), cytomegalovirus (CMV, such as TCN-202 is in development by Theraclone Sciences), human herpesvirus 6 (HHV-6), B virus (herpesvirus simiae), Flavivirus encephalitis, Japanese encephalitis, Murray valley fever, JC virus (progressive multifocal leukoencephalopathy), Nipah Virus (NiV), measles (subacute sclerosing panencephalitis); and other infections, such as, e.g., subactuate sclerosing panencephalitis, progressive multifocal leukoencephalopathy; human immunodeficiency virus (acquired immunodeficiency syndrome (AIDS)); Streptococcus pyogenes and other .beta.-hemolytic Streptococcus (e.g., Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcal Infection, PANDAS) and/or Syndenham's chorea, and Guillain-Barre syndrome, and prions.

[0043] Examples of suitable antibody constructs may include those described, e.g., in WO 2007/012924A2, Jan. 29, 2015, which is incorporated by reference herein.

[0044] For example, other nucleic acid sequences may encode anti-prion immunoglobulin constructs. Such immunoglobulins may be directed against major prion protein (PrP, for prion protein or protease-resistant protein, also known as CD230 (cluster of differentiation 230). The amino acid sequence of PrP is provided, e.g., http://www.ncbi.nlm.nib.gov/protein/NP 000302, incorporated by reference herein. The protein can exist in multiple isoforms, the normal PrP.sup.C, the disease-causing PrP.sup.Sc, and an isoform located in mitochondria. The misfolded version PrP.sup.Sc is associated with a variety of cognitive disorders and neurodegenerative diseases such as Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, and kuru.

[0045] Once the target and immunoglobulin are selected, the coding sequences for the selected immunoglobulin (e.g. heavy and/or light chain(s)) may be obtained and/or synthesized. Methods for sequencing a protein, peptide, or polypeptide (e.g., as an immunoglobulin) are known to those of skill in the art. Once the sequence of a protein is known, there are web-based and commercially available computer programs, as well as service based companies which back translate the amino acids sequences to nucleic acid coding sequences. See, e.g., backtranseq by EMBOSS, http://www.ebi.ac.uk/Tools/st/; Gene Infinity (http://www.geneinfinity.org/sms/sms_backtranslation.html); ExPasy (http://www.expasy.org/tools/). In one embodiment, the RNA and/or cDNA coding sequences are designed for optimal expression in human cells.

[0046] Codon-optimized coding regions can be designed by various different methods. This optimization may be performed using methods which are available on-line (e.g., GeneArt), published methods, or a company which provides codon optimizing services, e.g., DNA2.0 (Menlo Park, Calif.). One codon optimizing algorithm is described, e.g., in US International Patent Publication No. WO 2015/012924, which is incorporated by reference herein. See also, e.g., US Patent Publication No. 2014/0032186 and US Patent Publication No. 2006/0136184. Suitably, the entire length of the open reading frame (ORF) for the product is modified. However, in some embodiments, only a fragment of the ORF may be altered. By using one of these methods, one can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide.

[0047] A number of options are available for performing the actual changes to the codons or for synthesizing the codon-optimized coding regions designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence are synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides are designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO.RTM. vector available from Invitrogen Corporation, Carlsbad, Calif. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.

[0048] The immunoglobulin genes described herein may be used to express the "wild-type", a published or commercially available, or other known constant immunoglobulin domains or can be engineered to decrease affinity for, or ablate, binding to the Fc binding site present on immunoglobulins. There are several different types of Fc receptors, which are classified based on the type of antibody that they recognize. As used herein, "FcRn" refers to the neonatal Fc receptor that binds IgG. It is similar in structure to MHC class I protein. In humans, it is encoded by the FCGRT gene. The Fc receptor is located on various cells types, including, e.g., the epithelial cells of the blood brain barrier. The term "FcRn binding domain" as used herein refers to a protein domain that directly or indirectly binds to the FcRn. The FcRn may be a mammalian FcRn. In further embodiments, the FcRn is a human FcRn. An FcRn binding domain binding directly to an FcRn is an antibody Fc region. Meanwhile, regions capable of binding to a polypeptide such as albumin or IgG, which has human FcRn-binding activity, can indirectly bind to human FcRn via albumin, IgG, or such. Thus, such a human FcRn-binding region may be a region that binds to a polypeptide having human FcRn-binding activity. The term "Fc region" as used herein refers to an FcRn-binding domain that directly binds to FcRn, a mammalian FcRn, or a human FcRn. In particular, an Fc region is an Fc region of an antibody. The Fc region may be a mammalian Fc region or more particularly a human Fc region. In particular, the Fc region may be located within the second and third constant domain of a human immunoglobulin (CH2 and CH3). Further, the Fc region may be the hinge of CH2 and CH3. In one embodiment, the immunoglobulin construct is an IgG. In a further embodiment, the Fc region is an Fc region of human IgG1. Other Ig isotypes can be used as well.

[0049] Methods and computer programs for preparing such alignments are available and well known to those of skill in the art. Substitutions may also be written as (amino acid identified by single letter code)-position #-(amino acid identified by single letter code) whereby the first amino acid is the substituted amino acid and the second amino acid is the substituting amino acid at the specified position. The terms "substitution" and "substitution of an amino acid" and "amino acid substitution" as used herein refer to a replacement of an amino acid in an amino acid sequence with another one, wherein the latter is different from the replaced amino acid. Methods for replacing an amino acid are well known to the skilled in the art and include, but are not limited to, mutations of the nucleotide sequence encoding the amino acid sequence. Methods of making amino acid substitutions in IgG are described, e.g., for WO 2013/046704, which is incorporated by reference for its discussion of amino acid modification techniques, although this document describes increasing FcRn affinity, rather than decreasing or ablating binding affinity as described herein.

[0050] The term "amino acid substitution" and its synonyms described above are intended to encompass modification of an amino acid sequence by replacement of an amino acid with another, substituting amino acid. The substitution may be a conservative substitution. The term conservative, in referring to two amino acids, is intended to mean that the amino acids share a common property recognized by one of skill in the art. The term non-conservative, in referring to two amino acids, is intended to mean that the amino acids which have differences in at least one property recognized by one of skill in the art. For example, such properties may include amino acids having hydrophobic nonacidic side chains, amino acids having hydrophobic side chains (which may be further differentiated as acidic or nonacidic), amino acids having aliphatic hydrophobic side chains, amino acids having aromatic hydrophobic side chains, amino acids with polar neutral side chains, amino acids with electrically charged side chains, amino acids with electrically charged acidic side chains, and amino acids with electrically charged basic side chains. Both naturally occurring and non-naturally occurring amino acids are known in the art and may be used as substituting amino acids in embodiments. Thus, a conservative amino acid substitution may involve changing a first amino acid having a hydrophobic side chain with a different amino acid having a hydrophobic side chain; whereas a non-conservative amino acid substitution may involve changing a first amino acid with an acidic hydrophobic side chain with a different amino acid having a different side chain, e.g., a basic hydrophobic side chain or a hydrophilic side chain. Still other conservative or non-conservative changes change be determined by one of skill in the art.

[0051] In still other embodiments, the substitution at a given position will be to an amino acid, or one of a group of amino acids, that will be apparent to one of skill in the art in order to accomplish an objective identified herein.

[0052] In one embodiment, an immunoglobulin construct as defined herein is engineered so that the native sequence located on the conserved region of the immunoglobulin Fc region is ablated to eliminate binding to the FcRn and to minimize or eliminate transport of the proteinaceous immunoglobulin constructs across the blood brain barrier (out of the CNS area) and into the systemic circulation. In one example, this may be accomplished by altering one or more amino acids of the FcRn-binding domain, e.g., by modification of the codon for the selected amino acid(s). See. e.g., U.S. Pat. No. 8,618,252 B2,

[0053] Additionally or alternatively, immunoglobulin constructs (e.g., Fc variants) are engineered to have enhanced effector function are selected. See. e.g., T. Matsushita, Korean J Hematol, 2011 September; 46(3): 148-150; U.S. Pat. No. 6,946,292.

[0054] The heavy chain amino acid numbering used herein to identify the location of the mutants is based on the EU numbering system [IMGT unique numbering, Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969); http://www.imgt.org/-IMGTScientificChart/-Numbering/Hu_IGHGnber.h- tml]] and refer to positions in an FcRn-binding domain, in particular in an Fc region. In a similar fashion, substitutions are indicated as for example "EU387R" or "EU440E", wherein the number given after "EU" indicates the position of the substitution according the EU numbering, and the letter after the number is the substituted amino acid given in the one letter code. Other numbering systems include, e.g., Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, C. Foeler. (1991) Sequences of Proteins of Immunological Interest. No. 91-3242 U. S. Public Health Services, National Institutes of Health, Bethesda).

[0055] The promoter(s) can be selected from different sources, e.g., human cytomegalovirus (CMV) immediate-early enhancer/promoter, the SV40 early enhancer/promoter, the JC polymovirus promoter, myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) promoters, herpes simplex virus (HSV-1) latency associated promoter (LAP), rouse sarcoma virus (RSV) long terminal repeat (LTR) promoter, neuron-specific promoter (NSE), platelet derived growth factor (PDGF) promoter, hSYN, melanin-concentrating hormone (MCH) promoter, CBA, matrix metalloprotein promoter (MPP), and the chicken beta-actin promoter.

[0056] In certain embodiments, the expression cassette described herein contains at least one internal ribosome binding site, i.e., an IRES, located between the coding regions of the heavy and light chains. Alternatively the heavy and light chain may be separated by a furin-2a self-cleaving peptide linker [see. e.g., Radcliffe and Mitrophanous, Gene Therapy (2004), 11, 1673-1674. The expression cassette may contain at least one enhancer, i.e., CMV enhancer. Still other enhancer elements may include, e.g., an apolipoprotein enhancer, a zebrafish enhancer, a GFAP enhancer element, and brain specific enhancers such as described in WO 2013/1555222, woodchuck post hepatitis post-transcriptional regulatory element. Additionally, or alternatively, other, e.g., the hybrid human cytomegalovirus (HCMV)-immediate early (IE)-PDGR promoter or other promoter--enhancer elements may be selected. To enhance expression the other elements can be introns (like Promega.TM. intron or chimeric chicken globin-human immunoglobulin intron).

[0057] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., any one of the modified ORFs provided herein when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). As another example, polynucleotide sequences can be compared using Fasta, a program in GCG Version 6.1. Fasta provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. For instance, percent sequence identity between nucleic acid sequences can be determined using Fasta with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) as provided in GCG Version 6.1, herein incorporated by reference. Generally, these programs are used at default settings, although one skilled in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program that provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. This definition also refers to, or can be applied to, the compliment of a sequence. The definition also includes sequences that have deletions and/or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and oftentimes over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the full-length of an amino acid or nucleic acid sequences.

[0058] Typically, when an alignment is prepared based upon an amino acid sequence, the alignment contains insertions and deletions which are so identified with respect to a reference AAV sequence and the numbering of the amino acid residues is based upon a reference scale provided for the alignment. However, any given AAV sequence may have fewer amino acid residues than the reference scale. In the present invention, when discussing the parental sequence, the term "the same position" or the "corresponding position" refers to the amino acid located at the same residue number in each of the sequences, with respect to the reference scale for the aligned sequences. However, when taken out of the alignment, each of the proteins may have these amino acids located at different residue numbers. Alignments are performed using any of a variety of publicly or commercially available Multiple Sequence Alignment Programs. Sequence alignment programs are available for amino acid sequences, e.g., the "Clustal X", "MAP", "PIMA", "MSA", "BLOCKMAKER", "MEME", and "Match-Box" programs. Generally, any of these programs are used at default settings, although one of skill in the art can alter these settings as needed. Alternatively, one of skill in the art can utilize another algorithm or computer program which provides at least the level of identity or alignment as that provided by the referenced algorithms and programs. See. e.g., J. D. Thomson et al, Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0059] In another embodiment, a modified immunoglobulin having its affinity for FcRn ablated and retaining physiologically effective activity is provided. One or more amino acid modifications may be selected to ablate functional binding to FcRn. In one embodiment, the mutation lowers the binding affinity of the immunoglobulin for FcRn to less than 10% of the native protein. Suitably, the immunoglobulins with these mutations bind substantially normally to all other Fc receptors. Once the amino acid sequence is selected, the nucleic acid sequences can be designed and/or the previously described sequences may be engineered as described above. These modifications are made by engineering the nucleic acid coding region using site directed mutagenesis or other genetic engineering techniques which are known to those of skill in the art.

[0060] In one embodiment, the immunoglobulin genes described herein are engineered into a genetic element (e.g., a plasmid) useful for generating AAV vectors which transfer the immunoglobulin construct sequences carried thereon. The examples herein and SEQ ID NO: 13, 14 and 15, respectively illustrate AAV vector genomes with three different scFV constructs directed to A.beta. targets. The selected vector may be delivered to an AAV packaging cell by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high velocity DNA-coated pellets, viral infection and protoplast fusion. Stable packaging cells can also be made. The methods used to make such constructs are known to those with skill in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See. e.g., Molecular Cloning: A Laboratory Manual, ed. Green and Sambrook, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2012).

AAV Vectors

[0061] An AAV vector as described herein can comprise one or more nucleic acid sequences, each of which encodes one or more of the heavy and/or light chain polypeptides, or other polypeptides, of an immunoglobulin construct. Suitably, a composition contains one or more AAV vectors which contain nucleic acid sequences encoding all of the polypeptides which form an immunoglobulin construct in vivo. For example, a full-length antibody consists of four polypeptides: two copies of a heavy (H) chain polypeptide and two copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. In this respect, an AAV vector as described herein can comprise a single nucleic acid sequence that encodes the heavy chain polypeptides (e.g., constant variable) and the light chain polypeptides of an immunoglobulin construct. Alternatively, the AAV vector can comprise a first expression cassette that encodes at least one heavy chain constant polypeptides and at least one heavy chain variable polypeptide, and a second expression cassettes that encodes constant and variable light chain polypeptides of an immunoglobulin construct. In yet another embodiment, the AAV vector can comprise a first expression cassette encoding a first heavy chain polypeptide, a second expression cassette encoding a second heavy chain polypeptide, and a third expression cassette encoding a light chain polypeptide is shared by the two heavy chains. In another embodiment, an AAV vector may express 1, 2, 3 or 4 scFv open reading frames (ORFs), each of which may be same or different.

[0062] Typically, an expression cassette for an AAV vector comprises an AAV 5' inverted terminal repeat (ITR), the immunoglobulin construct coding sequences and any regulatory sequences, and an AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, termed .DELTA.ITR, has been described in which the D-sequence and terminal resolution site (trs) are deleted. In other embodiments, the full-length AAV 5' and 3' ITRs are used.

[0063] Where a pseudotyped AAV is to be produced, the ITRs in the expression are selected from a source which differs from the AAV source of the capsid. For example, AAV2 ITRs may be selected for use with an AAV capsid having a particular efficiency for targeting CNS or tissues or cells within the CNS. In one embodiment, the ITR sequences from AAV2, or the deleted version thereof (.DELTA.ITR), are used for convenience and to accelerate regulatory approval. However, ITRs from other AAV sources may be selected. Where the source of the ITRs is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be termed pseudotyped. However, other sources of AAV ITRs may be utilized.

[0064] The abbreviation "scAAV" refers to self-complementary. "Self-complementary AAV" refers a construct in which a coding region carried by a recombinant AAV nucleic acid sequence has been designed to form an intra-molecular double-stranded DNA template. Upon infection, rather than waiting for cell mediated synthesis of the second strand, the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. See. e.g., D M McCarty et al, "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis", Gene Therapy, (August 2001), Vol 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described in, e.g., U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683, each of which is incorporated herein by reference in its entirety.

[0065] The expression cassette typically contains a promoter sequence as part of the expression control sequences, e.g., located between the selected 5' ITR sequence and the immunoglobulin construct coding sequence. Tissue specific promoters, constitutive promoters, regulatable promoters [see. e.g., WO 2011/126808 and WO 2013/04943], or a promoter responsive to physiologic cues may be used may be utilized in the vectors described herein. In addition to a promoter, an expression cassette and/or a vector may contain other appropriate transcription initiation, termination, enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.

[0066] These control sequences are "operably linked" to the immunoglobulin construct gene sequences. As used herein, the term "operably linked" refers to both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0067] In one embodiment, a self-complementary AAV is provided. This viral vector may contain a .DELTA.5' ITR and an AAV 3' ITR. In another embodiment, a single-stranded AAV viral vector is provided. Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, e.g., U.S. Pat. Nos. 7,790,449; 7,282,199; WO 2003/042397; WO 2005/033321, WO 2006/110689; and U.S. Pat. No. 7,588,772B2]. In one system, a producer cell line is transiently transfected with a construct that encodes the transgene flanked by ITRs and a construct(s) that encodes rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding the transgene flanked by ITRs. In each of these systems, AAV virions are produced in response to infection with helper adenovirus or herpesvirus, requiring the separation of the rAAVs from contaminating virus. More recently, systems have been developed that do not require infection with helper virus to recover the AAV--the required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied, in trans, by the system. In these newer systems, the helper functions can be supplied by transient transfection of the cells with constructs that encode the required helper functions, or the cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or posttranscriptional level. In yet another system, the transgene flanked by ITRs and rep/cap genes are introduced into insect cells by infection with baculovirus-based vectors. For reviews on these production systems, see generally, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of each of which is incorporated herein by reference in its entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which is incorporated herein by reference in its entirety: 5,139,941; 5,741,683; 6,057,152; 6,204,059; 6,268,213; 6,491,907; 6,660,514; 6,951,753; 7,094,604; 7,172,893; 7,201,898; 7,229,823; and 7,439,065.

[0068] The available space for packaging may be conserved by combining more than one transcription unit into a single expression cassette, thus reducing the amount of required regulatory sequences. For example, a single promoter may direct expression of a single RNA that encodes two or three or more genes, and translation of the downstream genes are driven by IRES sequences. In another example, a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three or more genes separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A) and/or a protease recognition site (e.g., furin). The ORF thus encodes a single polyprotein, which, either during or after translation, is cleaved into the individual proteins (such as, e.g., heavy chain and light chain). It should be noted, however, that although these IRES and polyprotein systems can be used to save AAV packaging space, they can only be used for expression of components that can be driven by the same promoter. In another alternative, the transgene capacity of AAV can be increased by providing AAV ITRs of two genomes that can anneal to form head to tail concatamers. In a further alternative, bidirectional promoters may be selected.

[0069] In the examples below, an AAV vector having an AAV9 capsid is described. As used herein, "AAV9 capsid" refers to the AAV9 having the amino acid sequence of GenBank accession:AAS99264, which is incorporated by reference herein. Some variation from this encoded sequence is encompassed by the present invention, which may include sequences having at least about 95%, at least about 97% or at least about 99% identity to the referenced amino acid sequence in GenBank accession:AAS99264 and U.S. Pat. No. 7,906,111 (also WO 2005/033321). Methods of generating the capsid, coding sequences therefore, and methods for production of rAAV viral vectors have been described. See, e.g., Gao, et al, Proc. Natl. Acad. Sci. U.S.A. 100 (10), 6081-6086 (2003) and US 2013/0045186A1. Generation of AAV9 vectors is described, e.g., in U.S. Pat. No. 7,906,111, which is incorporated herein by reference. However, other sources of AAV capsids and other viral elements may be selected, as may other immunoglobulin constructs and other vector elements. Methods of generating AAV vectors have been described extensively in the literature and patent documents, including, e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; U.S. Pat. No. 7,588,772 B2. The source of AAV capsids may be selected from an AAV which targets CNS, specific cells within the CNS, and/or specific antigens or receptors. Suitable AAV may include, e.g, AAV9 [U.S. Pat. No. 7,906,111; US 2011-0236353-A1], rh10 [WO 2003/042397] and/or hu37 [see, e.g., U.S. Pat. No. 7,906,111; US 2011-0236353-A1]. However, other AAV, including, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 [U.S. Pat. Nos. 7,790,449; 7,282,199] and others may be selected for preparing the AAV vectors described herein.

Uses and Regimens

[0070] Suitably, the composition of the invention are designed so that AAV vectors carry the nucleic acid expression cassettes encoding the immunoglobulin constructs and regulatory sequences which direct expression of the immunoglobulin thereof in the selected cell. Following administration of the vectors into the CNS, the vectors deliver the expression cassettes to the CNS and express the proteinaceous immunoglobulin constructs in vivo. The use of compositions described herein for a therapeutic purpose are described, as are uses of these compositions in various regimens, which may optionally involve delivery of one or more other active agents.

[0071] As stated above, a composition may contain a single type of AAV vector as described herein which contains the expression cassette for delivering the immunoglobulin construct in vivo. Alternatively, a composition may contain two or more different AAV vectors, each of which has packaged therein different expression cassettes. For example, the two or more different AAV may have different expression cassettes which express immunoglobulin polypeptides which assemble in vivo to form a single functional immunoglobulin construct. In another example, the two or more AAV may have different expression cassettes which express immunoglobulin polypeptides for different targets, e.g., two provide for two functional immunoglobulin constructs (e.g., a first immunoglobulin construct and a second immunoglobulin construct). In still another alternative, the two or more different AAV may express immunoglobulin constructs directed to the same target, wherein one of the immunoglobulin constructs has been modified to ablate FcRn binding and a second immunoglobulin construct which retains its ability or has enhanced ability to bind to FcRn. Such a composition may be useful to simultaneously provide antibodies with increased retention in the CNS and antibodies for systemic delivery of the immunoglobulin construct.

[0072] A regimen as described herein may comprise, in addition to one or more of the combinations described herein, further combination with one or more of a biological drug, a small molecule drug, or other therapy. A biological drug as described herein, is based on a peptide, polypeptide, protein, enzyme, nucleic acid molecule, vector (including viral vectors), or the like.

[0073] Suitably, the compositions described herein comprise an effective amount of one or more AAV suspended in a pharmaceutically suitable carrier and/or admixed with suitable excipients designed for delivery to the subject via injection, osmotic pump, intrathecal catheter, or for delivery by another device or route. In one example, the composition is formulated for intrathecal delivery. In one embodiment, intrathecal delivery encompasses an injection into the spinal canal, e.g., the subarachnoid space. However, other routes of delivery may be selected and the pharmaceutically acceptable carriers for the AAV compositions including, e.g., intracranial, intranasal, intracisternal, intracerebrospinal fluid delivery, among other suitable direct or systemic routes, i.e. Ommaya reservoir.

[0074] The compositions can be formulated to contain an amount of AAV that is in the range of about 1.times.10.sup.9 genome copies (GC) to about 5.times.10.sup.14 GC. In one example, the vector is about 3.times.10.sup.13GC, but other amounts such as about 1.times.10.sup.9 GC, about 5.times.10.sup.9 GC, about 1.times.10.sup.10 GC, about 5.times.10.sup.10 GC, about 1.times.10.sup.11 GC, about 5.times.10.sup.11GC, about 1.times.10.sup.11 GC, about 5.times.10.sup.11 GC, or about 5.times.10.sup.13GC. Such compositions may contain a single AAV stock which expresses an immunoglobulin directed to a selected target. In another embodiment, such compositions may contain two AAV stock which co-express an immunoglobulin which assembles in the targeted host cell to form the desired immunoglobulin (e.g., a full-length antibody) against a selected target. In another embodiment, a composition may contain two or more AAV stock, each of which expresses a different immunoglobulin construct. In such compositions, the expressed proteins may combine to form a single immunoglobulin or may express two or more immunoglobulins having different targets. These different targets may be to different ligands on the same cell type or the same virus (or other target), or to two completely different pathogens. The compositions are designed for intrathecal delivery. In one embodiment, a spinal tap is performed in which from about 15 mL (or less) to about 25 mL CSF is removed and in which vector is suspended in a compatible carrier and delivered to the subject.

[0075] The rAAV, preferably suspended in a physiologically compatible carrier and optionally admixed with one or more excipients, may be administered to a human or non-human mammalian patient. Suitable carriers may be readily selected by one of skill in the art in view of the indication for which the transfer virus is directed. For example, one suitable carrier includes saline, which may be formulated with a variety of buffering solutions (e.g., phosphate buffered saline). Other exemplary carriers include Elliot's B, sterile saline, lactose, sucrose, maltose, and water. The selection of the carrier is not a limitation of the present invention. Optionally, the compositions of the invention may contain, in addition to the rAAV and carrier(s), other conventional pharmaceutical ingredients, such as preservatives, or chemical stabilizers.

[0076] In one embodiment, the compositions described herein are used in preparing medicaments for treating central nervous system disorders and diseases.

[0077] In another aspect, a method for treatment of Alzheimer's Disease is provided which involves intrathecal delivery of an AAV vector composition as described herein, in which at least one AAV vector stock expresses an immunoglobulin specific for a A.beta., beta secretase, and/or the tau protein, to subject in need thereof.

[0078] In still another embodiment, a method for treatment of Parkinson's Disease or related synucleinopathies is provide which involves intrathecal delivery of an AAV vector composition as provided herein, in which at least one AAV vector stock encodes one or more leucine-rich repeat kinase 2 antibody, dardarin (LRRK2) antibody, alpha-synuclein antibody, and/or DJ-1 (PARK7) antibody.

[0079] In yet a further aspect, a method for treatment of multiple sclerosis is provided herein which involves intrathecal delivery of an AAV vector composition as provided herein, in which composition at least one vector stock encodes an immunoglobulin directed against one or more of an a4-integrin, CD20, CD25, IL 12, p40+IL23p40, LINGO-1, CD40, and rHIgM22, CD52, IL17, CD19, and/or SEMA4D.

[0080] In yet another aspect, a method for treatment of ALS is provided which involves intrathecal delivery of an AAV vector composition as described herein, in which at least one AAV vector stock expresses an immunoglobulin specific for ALS enzyme superoxide dismutase 1 (SOD1) and variants thereof, a Derlin-1-binding region, and/or an antibody construct against neurite outgrowth inhibitor. In one embodiment, the vector and/or composition comprises an anti-SOD1 immunoglobulin Fc fragment.

[0081] In one aspect a method is provided for treatment of prion related diseases which comprises intrathecal delivery of an AAV vector composition, in which at least one vector stock encodes an immunoglobulin directed one or more of major prion protein, or PrPSc.

[0082] Animal models for assessing the effectiveness of a treatment against a variety of the disorders and diseases described herein are available. See, e.g., animal models for assessing Alzheimer's disease described, e.g., in D Van Dam and P. P. De Deyn, Br J Pharmacol, 2011 October; 164(4): 1285-1300. Models for assessing Parkinson's disease drugs have also been described, e.g., in HT Tran et al, Cell Reports, Vol. 7, Issue 6, p2054-2065, Jun. 26, 2014., R M Ransajoff, Nature Neuroscience, August 2012, Vol 15, 1074-1077 (multiple sclerosis); M. A Pouladi et al, Natuer Reviews Neuroscience, 14, 708-721 (2013) (Huntingdon's disease); N Fernandez-Borges, et al., Cur top Med Chem 2013; 13(19): 2504-21 (anti-prion drugs); PMcGoldrick, et al, Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, Vol. 1832, Issue 9, September 2013, pp. 1421-1436 and J M Moser et al, Mol Genet Genomics, 2013 June; 288 (5-6): 207-29 (ALS). Still other models are known to those of skill in the art.

[0083] In another aspect, a method for treatment of infectious disease of the central nervous system is provided which involves intrathecal delivery of an AAV vector composition as provided herein, in which composition at least one vector stock encodes an immunoglobulin directed against the pathogen which causes said infectious disease. Examples, without limitation, include one or more immunoglobulins directed against one or more of Mycobacterium tuberculosis (tuberculosis), Neisseria meningitides (meningitis), Streptococcus pneumonia, Listeria monocytogens (listeriosis), Borrelia burdorferia (lyme disease), human deficiency virus (acquired immunodeficiency syndrome), a herpes family viruses, varicella zoster virus, Epstein-Barr virus (EBV), cytomegalovirus, and/or JC virus.

[0084] Optionally, the AAV compositions as described herein are administered in the absence of an additional extrinsic pharmacological or chemical agent, or other physical disruption of the blood brain barrier.

[0085] In a combination therapy, the AAV-delivered immunoglobulin construct described herein is administered before, during, or after commencing therapy with another agent, as well as any combination thereof, i.e., before and during, before and after, during and after, or before, during and after commencing therapy

[0086] The compositions described herein may be used in a regimen involving co-administration of other active agents. Any suitable method or route can be used to administer such other agents. Routes of administration include, for example, systemic, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration. Optionally, the AAV compositions described herein may also be administered by one of these routes.

[0087] The following examples are illustrative only and are not a limitation on the invention described herein.

EXAMPLES

Example 1: CNS Expression of AAV9-Mediated Delivery of anti-SIV immunoadhesin

[0088] To evaluate the potential for long-term antibody production in the CNS following IT AAV delivery, a vector encoding an immunoadhesin was administered to two nonhuman primates, and the concentration of the transgene product in CSF was evaluated by ELISA. The AAV 9 vector expressing a rhesus macaque derived immunoadhesin (201IA) was constructed as previously described for AAV8, except the CB promoter (chicken beta actin with cytomegalovirus enhancer) was used in place of the cytomegalovirus promoter [Greig J A, et al. (2014) Intramuscular Injection of AAV8 in Mice and Macaques Is Associated with Substantial Hepatic Targeting and Transgene Expression. PLoS ONE 9(11): e112268] and the AAV9 capsid substituted for AAV8.

[0089] Briefly, the codon-optimized nucleotide sequence for rhesus macaque anti-SIV mac251 gp120 IgG-201 (Glamann et al. J Virol. 1998; 74(15):7158-7163. doi:10.1128/JVI.74.15.7158-7163.2000.Updated.) immunoadhesin (201IA) was cloned into an AAV expression construct. The construct was flanked by AAV2 inverted terminal repeats and contained a CB7 promoter, a chimeric intron, and a rabbit globin polyadenylation sequence (pAAV.CB7.CI.201IA.rBG). The sequence of this plasmid was confirmed [SEQ ID NO: 1 (SEQ ID NO: 2 corresponds to encoded 201IA sequence)].

[0090] The rAAV9.CB7.CI.201IA.rBG vector (3.times.10.sup.12 GC/kg) was delivered into the cerebrospinal fluid (CSF) of two cynomolgus macaques (IDs 0411 and 9860) by suboccipital puncture. Serum and CSF were collected periodically following vector administration. The concentration of 201IA in CSF and serum were determined by ELISA as follows.

[0091] All procedures were conducted at room temperature unless indicated otherwise. Plates were washed with BioTek 405TS microplate washer using PBS+0.05% Tween-20. mac251 gp120 (Immune Technology Corp.) diluted to 2 .mu.g/mL in PBS was incubated overnight on Costar.RTM. 96-well EasyWash.TM. ELISA assay plates (Corning) at 4.degree. C. Plates were then blocked 201IA ELISA blocking buffer (PBS+5% heat-inactivated fetal bovine serum+1 mM EDTA+0.07% Tween-20). Diluted samples were added to plates and diluted 2-fold down the plate at least four times. Plates were incubated for 1 h at 37.degree. C. and blocked again in 201IA ELISA blocking buffer. Plates were then incubated with AffiniPure polyclonal goat anti-human IgG-biotin (Jackson ImmunoResearch Labs) diluted in PBS then with streptavidin-horseradish peroxidase (Abcam) diluted in PBS. 3,3',5,5'-tetramethylbenzidine (TMB) substrate was used to develop the plates. After stopping the colorimetric reaction with H.sub.2SO.sub.4, plates were read using a SpectraMax M3 (Molecular Devices) plate reader at 450 nm.

[0092] The results for each of the two animals are illustrated in FIGS. 1A and 1B, which show the concentration of 201IA in cerebrospinal fluid and serum, respectively from administration to day 400 post-dose. The concentration scale for CSF is presented in ng/mL, whereas expression levels in serum were measured in micrograms (.mu.g)/mL.

Example 2-I253A Mutation of 201IA to Abrogate FcRn Binding

[0093] A nucleotide sequence 768 bp in length complementary to the 201IA gene but containing a mutation corresponding to I253A or H453A of the heavy chain amino acid sequence (Kabat numbering) was obtained from GeneArt (Life Technologies). The sequence was flanked by Pst1 and BstZ171 restriction sites matching those in pAAV.CB7.CI.201IA.rBG. The mutated sequences were separately cloned into a pAAV.CB7.CI.201IA.rBG by restriction digest using the enzymes indicated (NEB) and ligation (TaKaRa Inc.) as described by the manufacturers. Sanger sequencing (GeneWiz) was used to confirm complementarity of pAAV.CB7.CI.201IA.rBG [SEQ ID NO: 1 (SEQ ID NO: 2 corresponds to encoded 201IA sequence)], pAAV.CB7.CI.201IA(I253A).rBG [SEQ ID NO: 3 (encoding SEQ ID NO:4)] and pAAV.CB7.CI.201IA(H435A).rBG [SEQ ID NO: 5 (encoding SEQ ID NO:6] on either side of the desired mutation.

[0094] AAV9 vectors are generated using these plasmids using the methods described in Example 1.

Example 3--Generation Exemplary AAV.scFv Constructs

[0095] Three illustrative scFv constructs were prepared. These constructs were designed to eliminate the Fc region while still binding to the full-length anti-amyloid beta (A.beta.). These constructs were designed to reduce the risk of amyloid related imaging abnormalities and/or to limit the exposure of vessels to high monoclonal antibody concentrations.

[0096] A. Aducanumab scFv

[0097] The full-length antibody has been described as targeting fibrillary A.beta. [Weiner et al, Nature Reviews, Immunology, 6: 404-416 (May 2006)]. To generate the scFv, the amino acid sequences of the variable heavy (aa 21-143 of SEQ ID NO: 8) and variable light domains (aa 159-265 of SEQ ID NO: 8) of a recombinant, fully human anti-A.beta. IgG1 mAb (aducanumab, Biogen) were identified based on the WHO-published sequence [International Nonproprietary Names for Pharmaceutical Substances (INN), WHO Drug Information, Vol 27, No. 4, 2013, p401-402; see, also WO 2014/089500A1], which were codon optimized while being reverse translated to a nucleic acid sequence. The heavy and light domains were connected with a nucleic acid sequence encoding Gly-Ser linker (GGGGSGGGGSGGGGS, aa 144-158 of SEQ ID NO: 8). A coding sequence for an IL-2 secretion signal peptide (aa 1-20 of SEQ ID NO: 8) was fused to the 5' end of the nucleic acid sequence, and a coding sequence for 6.times.His tag was placed on the 3' end of the nucleic acid to facilitate purification. These sequences designed by us were ordered from and synthesized by a commercial contract facility. The plasmid contains, among other plasmid elements, an AAV vector genome consisting of: SEQ ID NO: 13: a 5' AAV2-ITR, a CMV IE promoter, a CB promoter, a chicken beta-actin intron, the scFV sequence, a rabbit beta-globin polyA, and an AAV2-3' ITR. This plasmid was co-expressed with a plasmid expressing the AAV9 capsid in the packaging 293 cell line. Also co-expressed in the packaging 293 cell line were the AAV rep functions and adenovirus helper functions required to package the AAV genome containing the scFV coding sequence and other regulatory elements flanked by the AAV ITRs into the AAV9 capsid. The resulting recombinant vector has an AAV9 capsid and the packaged AAV vector genome [SEQ ID NO: 13], termed AAV9.CB7.CI.aducanumabscFv.rGB.

[0098] In the study described herein, vector yield as determined by digital droplet PCR (ddPCR) was 5.89.times.10.sup.13 genome copies (GC/mL). Vector lots were assessed using the ddPCR techniques for determining AAV vector genome titers by ddPCR in M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb. 14.

[0099] B. Crenezumab_scFv

[0100] Crenezumab is a recombinant humanized monoclonal antibody against human 1-40 and 1-42 A.beta. (also termed .beta.-amyloid) developed by AC Immune and licensed to Genentech. Crenezumab is also knows at MABT5102A. It is characterized by having HVR region sequences of FIG. 2 of WO 2015/120233A1, and sequences of SEQ ID NO: 2-9, therein. The antibody has been described as being capable of targeted the oligomeric, soluble, and fibrillary A.beta..

[0101] The crenezumab scFv was prepared as described above for aducanumab, substituting the amino acid sequences of the variable heavy [aa 21-131 of SEQ ID NO: 10] and variable light chains [aa 147-258 of SEQ ID NO: 10] of crenezumab which were synthesized based on the WHO-published sequence [International Nonproprietary Names for Pharmaceutical Substances (INN), WHO Drug Information, Vol 25, No. 2, pp. 163-164 (2011); see, also WO 2015/120233A]. The resulting recombinant vector has an AAV9 capsid and the packaged AAV vector genome [SEQ ID NO: 141, termed AAV9.CB7.CI.crenezumabscFv.rGB.

[0102] In the study described herein, vector yield as determined by ddPCR was 3.79.times.10.sup.1 GC/mL. Vector lots were purified and assessed using ddPCR as described, M. Lock et al, Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 April; 25(2):115-25. doi: 10.1089/hgtb.2013.131. Epub 2014 Feb. 14.

[0103] C. Solanezumab scFv Solanezumab is a recombinant humanized monoclonal antibody available from Eli Lilly. Although it has been described as being highly homologous to crenezumab, it targets only the soluble A.beta., whereas crenezumab targets the oligomeric, soluble and fibrillar A.beta.. Solanezumab is characterized by having HVR region sequences of U.S. Pat. No. 7,195,761. The sequences used to construct the scFV was obtained from International Nonproprietary Names for Pharmaceutical Substances (INN), WHO Drug Information, Vol. 23, No. 3, pp. 263-264 (2009).

[0104] The solanezumab scFv was prepared as described above for aducanumab, substituting the amino acid sequences of the variable heavy [aa 21-131 of SEQ ID NO: 12] and variable light chains [aa 147-258 of SEQ ID NO: 12] of solanezumab which were synthesized based on the WHO-published sequence [International Nonproprietary Names for Pharmaceutical Substances (INN), WHO Drug Information, Vol 25, No. 2, pp. 163-164 (2011); see, also WO 2015/120233A]. The resulting recombinant vector has an AAV9 capsid and the packaged AAV vector genome [SEQ ID NO: 15], termed AAV9.CB7.CI.solanezumabscFv.rGB.

[0105] In the study described herein, ddPCR yield was 5.25.times.10.sup.13 GC/mL. Vector lots were purified and titered by ddPCR as described.

[0106] D. ELISA to Detect Anti-A.beta. scFvs

[0107] The scFv expression plasmids of Parts A-C above were used to produce the ELISA standards prior to packaging the plasmid in the AAV9 capsid. To purify the scFv ELISA standards, the pAAV plasmid were transfected into 293 cells. The scFvs were secreted into the cell culture supernatant. Taking advantage of the His-tag on the end of the scFvs, the scFvs were purified by running the supernatant over a His-trap column [GE Healthcare]. The remainder of the supernatant flows through the column. The bound His-tagged material (scFvs) was then eluted using a high-salt buffer.

[0108] The following enzyme linked immunsorbent assay (ELISA) was used to detect anti-A.beta. scFvs. The plate is coated with 1 .mu.g/mL recombinant human amyloid beta at 4.degree. C. overnight, using the "mixed-species" amyloid (aggregated and monomeric forms of the peptide). The assay is performed as follows. Wash 5.times. with PBS+0.05% Tween-20. Block with PBS+1% bovine serum albumin for 1 hour at room temperature. Add serum/brain lysate/purified anti-A.beta. scFvs. The three scFvs described in Parts A, B and C of this example were constructed with a His tag to facilitate the ELISA analysis, and they were purified from 293 supernatant using a His-trap column [His-Trap FF 1 ml column, GE Healthcare] according to manufacturer's instructions [http://www.gelifesciences.com/-webapp/wcs/stores/servlet/catalog/en/GELi- feSciencesus/products/AlternativeProductStructur e_17555/17524701]. Incubate at 37.degree. C. for one hour. Wash 5.times. with PBS+0.05% Tween-20. Add anti-His tag antibody (AbCam ab1187) diluted 1:10,000 in PBS. Incubate at RT for one hour. Wash 5.times. with PBS+0.05% Tween-20. Develop with TMB substrate (3,3',5,5'-tetramethylbenzidine) for 30 minutes at RT. Add 2N H.sub.2SO.sub.4 to stop reaction. Read at 450 nm and 540 nm on plate reader.

[0109] E. Pilot Study for Anti-A.beta. scFvs in 3.times.TG Mouse Model

[0110] AAV9.CB7.aducanumabSCFV.RBG, AAV9.CB7.crenezumabSCFV.RBG, AAV9.CB7.solanezumabSCFV.RBG, or PBS are administered intracerebroventricular (ICV) to 6 week-old 3.times.TG mice (MMRRC Repository/Jackson) [See. e.g., R. Stemicdzuk, et al, "Characterization of the 3.times.Tg-AD mouse model of Alzheimer's disease: part 2. Behavioral and cognitive changes". Brain Res. 2010 Aug. 12; 1348:149-55. doi: 10.1016/j.brainres.2010.06.011. Epub 2010 Jun. 15 and R. Sterniczuk, et al, "Characterization of the 3.times.Tg-A.beta. mouse model of Alzheimer's disease: part 1. Circadian changes.", Brain Res. 2010 Aug. 12; 1348:139-48. doi: 10.1016/j.brainres.2010.05.013. Epub 2010 May 31.] at 1.times.10.sup.11 gc/mouse, 6 mice per dosing cohort. Control mice (do not develop Alzheimer's pathology): B6129SF2/J (Jackson). Serum is taken monthly to monitor scFv expression. Mice undergo Morris water maze and/or Y-maze alternation behavioral testing at 6 months post-vector administration [See, e.g., Webster et al. Using mice to model Alzheimer's dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models. Front Genet. 2014; 5: 88.]. The Morris water maze is designed to measure spatial memory, movement control and cognitive mapping [Whishaw, I. Q. (1995). "A comparison of rats and mice in a swimming pool place task and matching to place task: some surprising differences". Physiology & Behavior. 58 (4): 687-693. doi:10.1016/0031-9384(95)00110-5; Crusio, Wim (1999). "Methodological considerations for testing learning in mice". In Crusio, W. E.; Gerlai, R.T. Handbook of molecular-genetic techniques for brain and behavior research (1st ed.). Amsterdam: Elsevier. pp. 638-651.j. The Y maze is used for assessing spatial working memory in rats and mice, especially for spontaneous alternation tasks. The maze may be purchases commercially, e.g., from Panlab. Mice are euthanized at 12 months of age for histological evaluation of A.beta. pathology and quantification of scFv/amyloid by ELISA from brain lysate. Other preliminary studies examine the efficacy of scFvs in older mice (dosing at .about.4-6 months, which is the average age of onset of visible A.beta. pathology in this model). Cohorts of mice are dosed at 6 weeks of age and at 4-6 months of age for histological evaluation of tau protein pathology, followed by euthanizing the mice at 15 months of age.

[0111] This application contains sequences and a sequence listing, which is hereby incorporated by reference. All publications, patents, and patent applications, including priority U.S. Patent application 62/247,498, filed Oct. 28, 2015, cited in this application are hereby incorporated by reference in their entireties as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

TABLE-US-00001 TABLE (Sequence Listing Free Text) The following information is provided for sequences containing free text under numeric identifier <223>. SEQ ID NO: (containing free text) Free text under <223> 1 <220> <221> misc feature <222> (275) . . . (404) <223> 3' ITR (complement) <220> <221> misc_feature <222> (3226) . . . (3355) <223> 5' ITR <220> <221> misc <222> (3423) . . . (3804) <223> CMV IE promoter <220> <221> CDS <222> (5161) . . . (6690) <223> 201IA 3 <220> <221> misc <222> (1) . . . (130) <223> 5' ITR <220> <221> misc_feature <222> (198) . . . (579) <223> CMV IE promoter <220> <221> promoter <222> (582) . . . (863) <223> CB promoter <220> <221> Intron <222> (958) . . . (1930) <223> chicken beta-actin intron <220> <221> CDS <222> (1936) . . . (3465) <223> CMV IE promoter <220> <221> polyA_signal <222> (3529) . . . (3655) <223> rabbit globin polyA <220> <221> misc_feature <222> (3744) . . . (3873) <223> 3' ITR (complement) <220> <221> misc_feature <222> (4636) . . . (5493) <223> AP(R) marker 4 <223> Synthetic Construct 5 <221> misc_feature <222> (275) . . . (404) <223> 3' ITR (complement) <220> <221> misc_feature <222> (3423) . . . (3804) <223> CMV IE promoter <220> <221> promoter <222> (3807) . . . (4088) <223> CB promoter <220> <221> TATA_signal <222> (4061) . . . (4064) <223> rabbit globin polyA <220> <221> Intron <222> (4183) . . . (5155) <223> chicken beta-actin intron <220> <221> CDS <222> (5161) . . . (6690) <223> CMV IE promoter 6 <220> <223> Synthetic Construct 7 <223> engineered aducanumab scFv coding sequence 8 <223> Aducanumab scFv construct amino acid sequence <220> <221> SIGNAL <222> (1) . . . (20) <223> IL2 secretion signal <220> <221> MISC_FEATURE <222> (21) . . . (143) <220> <221> MISC_FEATURE <222> (144) . . . (158) <223> Gly-Ser Llinker <220> <221> MISC FEATURE <222> (159) . . . (265) <223> Light Variable region <220> <221> MISC_FEATURE <222> (266) . . . (271) <223> 6xHistag (Explasy) 9 <223> Engineered crenezumab scFv construct 10 <223> Engineered Crenezumab scFv amino acid sequence <220> <221> SIGNAL <222> (1) . . . (20) <223> IL-2 secretion signal <220> <221> MISC_FEATURE <222> (21) . . . (131) <223> Heavy variable region <220> <221> MISC_FEATURE <222> (131) . . . (146) <223> Gly-Ser Linker <220> <221> MISC_FEATURE <222> (147) . . . (258) <223> Light Variable Region <220> <221> MISC_FEATURE <222> (259) . . . (264) <223> 6xHis tag 11 <223> engineered Solanezumab scFv construct nucleic acid sequence: 12 <223> engineered Solanezumab scFv construct amino acid sequence <220> <221> SIGNAL <222> (1) . . . (20) <223> IL2 secretion signal <220> <221> MISC_FEATURE <222> (21) . . . (131) <223> Heavy chain variable <220> <221> MISC_FEATURE <222> (132) . . . (146) <220> <221> MISC_FEATURE <222> (147) . . . (258) <223> Variable Light Region <220> <221> MISC_FEATURE <222> (259) . . . (264) <223> 6xHis tag 13 <223> AAV vector genome CB7.CI.aducanumabscFvRBG <220> <221> repeat_region <222> (1) . . . (130) <223> 5'' AAV2-ITR <220> <221> repeat_region <222> (198) . . . (579) <223> CMV IE promoter <220> <221> promoter <222> (582) . . . (863) <223> CB promoter <220> <221> Intron <222> (958) . . . (1930) <223> chicken beta actin promoter <220> <221> misc_feature <222> (2002) . . . (2370) <223> aducanumab heavy variable region <220> <221> misc_feature <222> (2371) . . . (2415) <223> Gly-Ser Linker <220> <221> misc_feature <222> (2416) . . . (2736) <223> aducanumab light chain variable <220> <221> misc_feature <222> (2737) . . . (2754) <223> His Tag <220> <221> polyA_signal <222> (2821) . . . (2947) <223> Rabbit globulin polya <220> <221> repeat_region <222> (3036) . . . (3165) <223> 3' ITR 14 <223> pAAV.CB7.CI.crenezumabScFv.RBG <220> <221> repeat_region <222> (1) . . . (130) <223> AAV2 - 5' ITR <220> <221> repeat_region <222> (198) . . . (579) <223> CMV IE promoter <220> <221> promoter <222> (582) . . . (863) <223> CB promoter <220> <221> misc_feature <222> (1942) . . . (2001) <223> IL-2 secretion signal <220> <221> misc_feature <222> (2001) . . . (2334) <223> crenezumab heavy variable region <220> <221> misc_feature <222> (2335) . . . (2379) <223> Gly-Ser linker <220> <221> misc_feature <222> (2380) . . . (2715) <223> crenezumab varalbe region light <220> <221> misc_feature <222> (2716) . . . (2733) <223> His tag <223> rabbit globin polyA <220> <221> repeat_region <222> (3015) . . . (3144) <223> AAV2 - 3' ITR

15 <223> pAAV.CB7.CI.solanezumabscFv.RBG <220> <221> repeat_region <222> (1) . . . (130) <223> AAV2 - 5' ITR <220> <221> repeat_region <222> (198) . . . (579) <223> CMV IE promoter <220> <221> promoter <222> (582) . . . (863) <223> CB promoter <220> <221> Intron <222> (958) . . . (1930) <223> chicken beta-actin intron <220> <221> misc_feature <222> (1942) . . . (2001) <223> IL-2 secretion signal <220> <221> misc_feature <222> (2002) . . . (2334) <223> solanezumab heavy variable region <220> <221> misc_feature <222> (2335) . . . (2379) <223> Gly-Ser Linker <220> <221> miscjeature <222> (2380) . . . (2715) <223> solanezumab light chain variable region <220> <221> polyA_signal <222> (2800) . . . (2926) <223> rabbit globin polyA <220> <221> misc_feature <222> (3015) . . . (3144) <223> AAV2 - 3' ITR

Sequence CWU 1

1

1516694DNAArtificial sequencepAAV.CB7.CI.201IA.rBGpolyA_signal(60)..(186)misc_feature(275)..(4- 04)3' ITR (complement)rep_origin(581)..(1036)misc_feature(3226)..(3355)5' ITRmisc(3423)..(3804)CMV IE promoterpromoter(3807)..(4088)TATA_signal(4061)..(4064)Intron(4183)..(515- 5)CDS(5161)..(6690)201IA 1gtacctctag agtcgacccg ggcggcctcg aggacggggt gaactacgcc tgaggatccg 60atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt 120ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 180cactcggaag caattcgttg atctgaattt cgaccaccca taatacccat taccctggta 240gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 300actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 360ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagccttaa ttaacctaat 420tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 480cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 540cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca 600ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 660gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 720caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 780cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 840tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 900acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 960gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 1020ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1080tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 1140ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 1200ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 1260aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 1320gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 1380ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 1440gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1500cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1560cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1620acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1680caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1740taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1800ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1860aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1920agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1980atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 2040tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 2100tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 2160gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 2220taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 2280aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata 2340ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 2400catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 2460ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2520ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2580agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2640taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2700atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2760cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2820ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2880accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2940gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 3000gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 3060agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 3120tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 3180gctatgacca tgattacgcc agatttaatt aaggccttaa ttaggctgcg cgctcgctcg 3240ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 3300gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 3360ttaatgatta acccgccatg ctacttatct accagggtaa tggggatcct ctagaactat 3420agctagtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 3480tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 3540accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 3600aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc 3660agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 3720gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 3780ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 3840ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 3900tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga 3960ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg 4020aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg 4080gcgggcgggg agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg 4140cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc 4200ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg 4260ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg 4320ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc 4380tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc 4440gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg 4500cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc 4560cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt 4620acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg 4680ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg 4740agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc 4800gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc 4860cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg 4920ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg 4980ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc 5040tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc 5100ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc 5160atg gag ttc ggg ctg agc tgg gtc ttt ctg gtg gcc ctg ctg aag gga 5208Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Lys Gly1 5 10 15gtc cag tgc gag gtg cag ctg ctg gaa tcc gga cct ggc ctg gtg aaa 5256Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys 20 25 30cca tct gag aca ctg agt ctg act tgt gct gtc tcc ggc ctg tct atc 5304Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Leu Ser Ile 35 40 45agc tcc gat ttc tcc tgg gca tgg att agg cag acc ccc ggc aag gcc 5352Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr Pro Gly Lys Ala 50 55 60ctg gaa tat gtg ggg tac atc cgc ggg aac acc gga gat aca tac tat 5400Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly Asp Thr Tyr Tyr65 70 75 80aat cct agt ctg aag tca agg ctg act atc tca aag gac acc agc aaa 5448Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95aac caa atc tac ctg aat ctg tct agt gtc acc gct ggc gat gcc gcc 5496Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala Gly Asp Ala Ala 100 105 110gtg tac tat tgc gca agg gac cgg gtg tgc gac gat gac tac gga tac 5544Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp Asp Tyr Gly Tyr 115 120 125tat tac acc gag gtg tgc ttc ggc ctg gat tct tgg ggg cag gga atc 5592Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp Gly Gln Gly Ile 130 135 140gtg gtc aca gtg tca agc ggc gga gga ggc agc gga gga gga ggg tcc 5640Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160gga ggc ggg gga tct gca gaa ctg gtc atg aca cag tcc cca ctg agc 5688Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln Ser Pro Leu Ser 165 170 175ctg tcc gtc gct cca gga cag act gca tct att agt tgt cga tcc tct 5736Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser Cys Arg Ser Ser 180 185 190cag tcc ctg gac tat gct aac ggc aat acc tac ctg tct tgg ttt cac 5784Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu Ser Trp Phe His 195 200 205cag cga cca gga cag cca cct cgg aga ctg atc tat cag att tcc aac 5832Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr Gln Ile Ser Asn 210 215 220aga gat tct gga gtg ccc gac agg ttc tca ggc agc gga gca gga act 5880Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr225 230 235 240gag ttt acc ctg cga atc agt cgg atg gaa tca gat gac gtg ggg atc 5928Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp Asp Val Gly Ile 245 250 255tac tac tgc gga cag ggg acc aca ttc cca cgg aca ttt gga cag ggc 5976Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr Phe Gly Gln Gly 260 265 270act aag gtg gag atc aaa acc tgt gga gga gga agc aag cca cca acc 6024Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr 275 280 285tgc cct cca tgt aca tct ccc gaa ctg ctg ggc ggg cct agc gtg ttc 6072Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 290 295 300ctg ttt ccc cct aag cct aaa gat aca ctg atg att agt aga acc cca 6120Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro305 310 315 320gag gtc aca tgc gtg gtc gtg gac gtg tcc cag gaa gat cct gac gtg 6168Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Asp Val 325 330 335aag ttc aac tgg tac gtg aat ggc gcc gag gtg cac cat gct cag act 6216Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His Ala Gln Thr 340 345 350aaa cca cgc gaa acc cag tat aat agt aca tac cga gtc gtg tca gtc 6264Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365ctg aca gtg act cac cag gat tgg ctg aac ggc aag gag tat acc tgc 6312Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys 370 375 380aag gtg tct aac aag gcc ctg ccc gcc cct atc cag aaa aca att agc 6360Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln Lys Thr Ile Ser385 390 395 400aag gac aaa ggg cag cca cgg gaa ccc cag gtg tac act ctg cca ccc 6408Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415tca aga gag gaa ctg act aag aac cag gtc agc ctg acc tgt ctg gtg 6456Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 420 425 430aaa ggc ttc tac ccc agc gat atc gtc gtg gag tgg gaa agt tca ggc 6504Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Ser Gly 435 440 445cag cct gag aat act tac aag act acc cct cca gtg ctg gat agc gac 6552Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460ggg tcc tat ttc ctg tac agc aag ctg aca gtg gac aaa tcc cgc tgg 6600Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp465 470 475 480cag cag gga aac gtc ttt tcc tgt tct gtg atg cat gag gcc ctg cac 6648Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495aat cat tac acc cag aag agt ctg tca ctg agc ccc ggc aaa tgag 6694Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 5102510PRTArtificial sequenceSynthetic Construct 2Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Leu Ser Ile 35 40 45Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr Pro Gly Lys Ala 50 55 60Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly Asp Thr Tyr Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala Gly Asp Ala Ala 100 105 110Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp Asp Tyr Gly Tyr 115 120 125Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp Gly Gln Gly Ile 130 135 140Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln Ser Pro Leu Ser 165 170 175Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser Cys Arg Ser Ser 180 185 190Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu Ser Trp Phe His 195 200 205Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr Gln Ile Ser Asn 210 215 220Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr225 230 235 240Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp Asp Val Gly Ile 245 250 255Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr Phe Gly Gln Gly 260 265 270Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr 275 280 285Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 290 295 300Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro305 310 315 320Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Asp Val 325 330 335Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His Ala Gln Thr 340 345 350Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys 370 375 380Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln Lys Thr Ile Ser385 390 395 400Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 420 425 430Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Ser Gly 435 440 445Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp465 470 475 480Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 51036694DNAArtificial sequencePlasmid encoding 201IA(I253A)mutantmisc(1)..(130)5' ITRmisc_feature(198)..(579)CMV IE promoterpromoter(582)..(863)CB promoterTATA_signal(836)..(839)Intron(958)..(1930)chicken beta-actin intronCDS(1936)..(3465)CMV IE promoterpolyA_signal(3529)..(3655)rabbit globin polyAmisc_feature(3744)..(3873)3' ITR (complement)rep_origin(4050)..(4505)misc_feature(4636)..(5493)AP(R) markerrep_origin(5667)..(6255) 3ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta 420aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960agcgggcggg acggcccttc tcctccgggc

tgtaattagc gcttggttta atgacggctt 1020gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg 1500cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920tttggcaaag aattc atg gag ttc ggg ctg agc tgg gtc ttt ctg gtg gcc 1971 Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala 1 5 10ctg ctg aag gga gtc cag tgc gag gtg cag ctg ctg gaa tcc gga cct 2019Leu Leu Lys Gly Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro 15 20 25ggc ctg gtg aaa cca tct gag aca ctg agt ctg act tgt gct gtc tcc 2067Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser 30 35 40ggc ctg tct atc agc tcc gat ttc tcc tgg gca tgg att agg cag acc 2115Gly Leu Ser Ile Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr45 50 55 60ccc ggc aag gcc ctg gaa tat gtg ggg tac atc cgc ggg aac acc gga 2163Pro Gly Lys Ala Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly 65 70 75gat aca tac tat aat cct agt ctg aag tca agg ctg act atc tca aag 2211Asp Thr Tyr Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys 80 85 90gac acc agc aaa aac caa atc tac ctg aat ctg tct agt gtc acc gct 2259Asp Thr Ser Lys Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala 95 100 105ggc gat gcc gcc gtg tac tat tgc gca agg gac cgg gtg tgc gac gat 2307Gly Asp Ala Ala Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp 110 115 120gac tac gga tac tat tac acc gag gtg tgc ttc ggc ctg gat tct tgg 2355Asp Tyr Gly Tyr Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp125 130 135 140ggg cag gga atc gtg gtc aca gtg tca agc ggc gga gga ggc agc gga 2403Gly Gln Gly Ile Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly 145 150 155gga gga ggg tcc gga ggc ggg gga tct gca gaa ctg gtc atg aca cag 2451Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln 160 165 170tcc cca ctg agc ctg tcc gtc gct cca gga cag act gca tct att agt 2499Ser Pro Leu Ser Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser 175 180 185tgt cga tcc tct cag tcc ctg gac tat gct aac ggc aat acc tac ctg 2547Cys Arg Ser Ser Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu 190 195 200tct tgg ttt cac cag cga cca gga cag cca cct cgg aga ctg atc tat 2595Ser Trp Phe His Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr205 210 215 220cag att tcc aac aga gat tct gga gtg ccc gac agg ttc tca ggc agc 2643Gln Ile Ser Asn Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser 225 230 235gga gca gga act gag ttt acc ctg cga atc agt cgg atg gaa tca gat 2691Gly Ala Gly Thr Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp 240 245 250gac gtg ggg atc tac tac tgc gga cag ggg acc aca ttc cca cgg aca 2739Asp Val Gly Ile Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr 255 260 265ttt gga cag ggc act aag gtg gag atc aaa acc tgt gga gga gga agc 2787Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser 270 275 280aag cca cca acc tgc cct cca tgt aca tct ccc gaa ctg ctg ggc ggg 2835Lys Pro Pro Thr Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly285 290 295 300cct agc gtg ttc ctg ttt ccc cct aag cct aaa gat aca ctg atg gcc 2883Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala 305 310 315agt aga acc cca gag gtc aca tgc gtg gtc gtg gac gtg tcc cag gaa 2931Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 320 325 330gat cct gac gtg aag ttc aac tgg tac gtg aat ggc gcc gag gtg cac 2979Asp Pro Asp Val Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His 335 340 345cat gct cag act aaa cca cgc gaa acc cag tat aat agt aca tac cga 3027His Ala Gln Thr Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg 350 355 360gtc gtg tca gtc ctg aca gtg act cac cag gat tgg ctg aac ggc aag 3075Val Val Ser Val Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys365 370 375 380gag tat acc tgc aag gtg tct aac aag gcc ctg ccc gcc cct atc cag 3123Glu Tyr Thr Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln 385 390 395aaa aca att agc aag gac aaa ggg cag cca cgg gaa ccc cag gtg tac 3171Lys Thr Ile Ser Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 400 405 410act ctg cca ccc tca aga gag gaa ctg act aag aac cag gtc agc ctg 3219Thr Leu Pro Pro Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu 415 420 425acc tgt ctg gtg aaa ggc ttc tac ccc agc gat atc gtc gtg gag tgg 3267Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp 430 435 440gaa agt tca ggc cag cct gag aat act tac aag act acc cct cca gtg 3315Glu Ser Ser Gly Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val445 450 455 460ctg gat agc gac ggg tcc tat ttc ctg tac agc aag ctg aca gtg gac 3363Leu Asp Ser Asp Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp 465 470 475aaa tcc cgc tgg cag cag gga aac gtc ttt tcc tgt tct gtg atg cat 3411Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 480 485 490gag gcc ctg cac aat cat tac acc cag aag agt ctg tca ctg agc ccc 3459Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 495 500 505ggc aaa tgaggtacct ctagagtcga cccgggcggc ctcgaggacg gggtgaacta 3515Gly Lys 510cgcctgagga tccgatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct 3575tgagcatctg acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa 3635ttttttgtgt ctctcactcg gaagcaattc gttgatctga atttcgacca cccataatac 3695ccattaccct ggtagataag tagcatggcg ggttaatcat taactacaag gaacccctag 3755tgatggagtt ggccactccc tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa 3815aggtcgcccg acgcccgggc tttgcccggg cggcctcagt gagcgagcga gcgcgcagcc 3875ttaattaacc taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 3935ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 3995aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc 4055cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac 4115ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg 4175ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt 4235tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc 4295cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct 4355tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga 4415ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga 4475attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg 4535aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 4595accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 4655tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 4715gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 4775ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 4835gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga 4895gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 4955agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 5015gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 5075cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct 5135gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 5195gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 5255ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 5315gtttattgct gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact 5375ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 5435tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 5495actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 5555taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga 5615gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 5675tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 5735ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 5795gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc 5855tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 5915cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 5975gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 6035actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 6095ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg 6155gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 6215atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 6275tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 6335tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 6395aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 6455gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 6515gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 6575ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 6635tcacacagga aacagctatg accatgatta cgccagattt aattaaggcc ttaattagg 66944510PRTArtificial sequenceSynthetic Construct 4Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Leu Ser Ile 35 40 45Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr Pro Gly Lys Ala 50 55 60Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly Asp Thr Tyr Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala Gly Asp Ala Ala 100 105 110Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp Asp Tyr Gly Tyr 115 120 125Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp Gly Gln Gly Ile 130 135 140Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln Ser Pro Leu Ser 165 170 175Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser Cys Arg Ser Ser 180 185 190Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu Ser Trp Phe His 195 200 205Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr Gln Ile Ser Asn 210 215 220Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr225 230 235 240Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp Asp Val Gly Ile 245 250 255Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr Phe Gly Gln Gly 260 265 270Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr 275 280 285Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 290 295 300Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ala Ser Arg Thr Pro305 310 315 320Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Asp Val 325 330 335Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His Ala Gln Thr 340 345 350Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys 370 375 380Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln Lys Thr Ile Ser385 390 395 400Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 420 425 430Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Ser Gly 435 440 445Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp465 470 475 480Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 51056694DNAArtificial sequenceengineered plasmid containining 201IA(H435) mutantmisc_feature(275)..(404)3' ITR (complement)rep_origin(2198)..(2786)misc_feature(3226)..(3355)misc_featur- e(3423)..(3804)CMV IE promoterpromoter(3807)..(4088)CB promoterTATA_signal(4061)..(4064)rabbit globin polyAIntron(4183)..(5155)chicken beta-actin intronCDS(5161)..(6690)CMV IE promoter 5gtacctctag agtcgacccg ggcggcctcg aggacggggt gaactacgcc tgaggatccg 60atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt 120ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct 180cactcggaag caattcgttg atctgaattt cgaccaccca taatacccat taccctggta 240gataagtagc atggcgggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 300actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 360ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagccttaa ttaacctaat 420tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 480cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat 540cgcccttccc aacagttgcg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca 600ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta 660gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt 720caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac 780cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt 840tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga 900acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg 960gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata 1020ttaacgctta caatttaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt 1080tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc 1140ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc 1200ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa 1260aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg 1320gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag 1380ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc 1440gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta 1500cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg 1560cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca 1620acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac 1680caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat 1740taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg 1800ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata 1860aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta 1920agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa 1980atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag 2040tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg 2100tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact 2160gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg 2220taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc 2280aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag

ataccaaata 2340ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta 2400catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc 2460ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg 2520ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac 2580agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg 2640taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt 2700atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct 2760cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg 2820ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata 2880accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca 2940gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc 3000gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg 3060agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta 3120tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca 3180gctatgacca tgattacgcc agatttaatt aaggccttaa ttaggctgcg cgctcgctcg 3240ctcactgagg ccgcccgggc aaagcccggg cgtcgggcga cctttggtcg cccggcctca 3300gtgagcgagc gagcgcgcag agagggagtg gccaactcca tcactagggg ttccttgtag 3360ttaatgatta acccgccatg ctacttatct accagggtaa tggggatcct ctagaactat 3420agctagtcga cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt 3480tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg 3540accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc 3600aatagggact ttccattgac gtcaatgggt ggactattta cggtaaactg cccacttggc 3660agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg 3720gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat 3780ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct gcttcactct 3840ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt aattattttg 3900tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg ggcggggcga 3960ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg gcgcgctccg 4020aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc gaagcgcgcg 4080gcgggcgggg agtcgctgcg acgctgcctt cgccccgtgc cccgctccgc cgccgcctcg 4140cgccgcccgc cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc 4200ccttctcctc cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg 4260ctgcgtgaaa gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg 4320ggtgcgtgcg tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc 4380tgtgagcgct gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc 4440gcggccgggg gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg 4500cggggtgtgt gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc 4560cccctgcacc cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt 4620acggggcgtg gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg 4680ggcggggcgg ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg 4740agcgccggcg gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc 4800gagagggcgc agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc 4860cgccgcaccc cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg 4920ggcggggagg gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg 4980ggctgtccgc ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc 5040tggcgtgtga ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc 5100ctacagctcc tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattc 5160atg gag ttc ggg ctg agc tgg gtc ttt ctg gtg gcc ctg ctg aag gga 5208Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Lys Gly1 5 10 15gtc cag tgc gag gtg cag ctg ctg gaa tcc gga cct ggc ctg gtg aaa 5256Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys 20 25 30cca tct gag aca ctg agt ctg act tgt gct gtc tcc ggc ctg tct atc 5304Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Leu Ser Ile 35 40 45agc tcc gat ttc tcc tgg gca tgg att agg cag acc ccc ggc aag gcc 5352Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr Pro Gly Lys Ala 50 55 60ctg gaa tat gtg ggg tac atc cgc ggg aac acc gga gat aca tac tat 5400Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly Asp Thr Tyr Tyr65 70 75 80aat cct agt ctg aag tca agg ctg act atc tca aag gac acc agc aaa 5448Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95aac caa atc tac ctg aat ctg tct agt gtc acc gct ggc gat gcc gcc 5496Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala Gly Asp Ala Ala 100 105 110gtg tac tat tgc gca agg gac cgg gtg tgc gac gat gac tac gga tac 5544Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp Asp Tyr Gly Tyr 115 120 125tat tac acc gag gtg tgc ttc ggc ctg gat tct tgg ggg cag gga atc 5592Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp Gly Gln Gly Ile 130 135 140gtg gtc aca gtg tca agc ggc gga gga ggc agc gga gga gga ggg tcc 5640Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160gga ggc ggg gga tct gca gaa ctg gtc atg aca cag tcc cca ctg agc 5688Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln Ser Pro Leu Ser 165 170 175ctg tcc gtc gct cca gga cag act gca tct att agt tgt cga tcc tct 5736Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser Cys Arg Ser Ser 180 185 190cag tcc ctg gac tat gct aac ggc aat acc tac ctg tct tgg ttt cac 5784Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu Ser Trp Phe His 195 200 205cag cga cca gga cag cca cct cgg aga ctg atc tat cag att tcc aac 5832Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr Gln Ile Ser Asn 210 215 220aga gat tct gga gtg ccc gac agg ttc tca ggc agc gga gca gga act 5880Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr225 230 235 240gag ttt acc ctg cga atc agt cgg atg gaa tca gat gac gtg ggg atc 5928Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp Asp Val Gly Ile 245 250 255tac tac tgc gga cag ggg acc aca ttc cca cgg aca ttt gga cag ggc 5976Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr Phe Gly Gln Gly 260 265 270act aag gtg gag atc aaa acc tgt gga gga gga agc aag cca cca acc 6024Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr 275 280 285tgc cct cca tgt aca tct ccc gaa ctg ctg ggc ggg cct agc gtg ttc 6072Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 290 295 300ctg ttt ccc cct aag cct aaa gat aca ctg atg att agt aga acc cca 6120Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro305 310 315 320gag gtc aca tgc gtg gtc gtg gac gtg tcc cag gaa gat cct gac gtg 6168Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Asp Val 325 330 335aag ttc aac tgg tac gtg aat ggc gcc gag gtg cac cat gct cag act 6216Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His Ala Gln Thr 340 345 350aaa cca cgc gaa acc cag tat aat agt aca tac cga gtc gtg tca gtc 6264Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365ctg aca gtg act cac cag gat tgg ctg aac ggc aag gag tat acc tgc 6312Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys 370 375 380aag gtg tct aac aag gcc ctg ccc gcc cct atc cag aaa aca att agc 6360Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln Lys Thr Ile Ser385 390 395 400aag gac aaa ggg cag cca cgg gaa ccc cag gtg tac act ctg cca ccc 6408Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415tca aga gag gaa ctg act aag aac cag gtc agc ctg acc tgt ctg gtg 6456Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 420 425 430aaa ggc ttc tac ccc agc gat atc gtc gtg gag tgg gaa agt tca ggc 6504Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Ser Gly 435 440 445cag cct gag aat act tac aag act acc cct cca gtg ctg gat agc gac 6552Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460ggg tcc tat ttc ctg tac agc aag ctg aca gtg gac aaa tcc cgc tgg 6600Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp465 470 475 480cag cag gga aac gtc ttt tcc tgt tct gtg atg cat gag gcc ctg cac 6648Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495aat gct tac acc cag aag agt ctg tca ctg agc ccc ggc aaa tgag 6694Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 5106510PRTArtificial sequenceSynthetic Construct 6Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Lys Gly1 5 10 15Val Gln Cys Glu Val Gln Leu Leu Glu Ser Gly Pro Gly Leu Val Lys 20 25 30Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Leu Ser Ile 35 40 45Ser Ser Asp Phe Ser Trp Ala Trp Ile Arg Gln Thr Pro Gly Lys Ala 50 55 60Leu Glu Tyr Val Gly Tyr Ile Arg Gly Asn Thr Gly Asp Thr Tyr Tyr65 70 75 80Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys 85 90 95Asn Gln Ile Tyr Leu Asn Leu Ser Ser Val Thr Ala Gly Asp Ala Ala 100 105 110Val Tyr Tyr Cys Ala Arg Asp Arg Val Cys Asp Asp Asp Tyr Gly Tyr 115 120 125Tyr Tyr Thr Glu Val Cys Phe Gly Leu Asp Ser Trp Gly Gln Gly Ile 130 135 140Val Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser145 150 155 160Gly Gly Gly Gly Ser Ala Glu Leu Val Met Thr Gln Ser Pro Leu Ser 165 170 175Leu Ser Val Ala Pro Gly Gln Thr Ala Ser Ile Ser Cys Arg Ser Ser 180 185 190Gln Ser Leu Asp Tyr Ala Asn Gly Asn Thr Tyr Leu Ser Trp Phe His 195 200 205Gln Arg Pro Gly Gln Pro Pro Arg Arg Leu Ile Tyr Gln Ile Ser Asn 210 215 220Arg Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr225 230 235 240Glu Phe Thr Leu Arg Ile Ser Arg Met Glu Ser Asp Asp Val Gly Ile 245 250 255Tyr Tyr Cys Gly Gln Gly Thr Thr Phe Pro Arg Thr Phe Gly Gln Gly 260 265 270Thr Lys Val Glu Ile Lys Thr Cys Gly Gly Gly Ser Lys Pro Pro Thr 275 280 285Cys Pro Pro Cys Thr Ser Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 290 295 300Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro305 310 315 320Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Asp Val 325 330 335Lys Phe Asn Trp Tyr Val Asn Gly Ala Glu Val His His Ala Gln Thr 340 345 350Lys Pro Arg Glu Thr Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 355 360 365Leu Thr Val Thr His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Thr Cys 370 375 380Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Gln Lys Thr Ile Ser385 390 395 400Lys Asp Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 405 410 415Ser Arg Glu Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 420 425 430Lys Gly Phe Tyr Pro Ser Asp Ile Val Val Glu Trp Glu Ser Ser Gly 435 440 445Gln Pro Glu Asn Thr Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 450 455 460Gly Ser Tyr Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp465 470 475 480Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 485 490 495Asn Ala Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 500 505 5107819DNAArtificial sequenceengineered aducanumab scFv coding sequence 7atgtacaaga tgcagctgct gagctgtatc gccctgaccc tggtgctggt ggccaactct 60caggtgcagc tggtggaatc tggcggcgga gtggtgcagc ctggcagaag cctgagactg 120agctgtgccg ccagcggctt cgccttcagc agctacggaa tgcactgggt gcgccaggcc 180cctggcaaag gactggaatg ggtggccgtg atttggttcg acggcaccaa gaagtactac 240accgacagcg tgaagggccg gttcaccatc agccgggaca acagcaagaa caccctgtac 300ctgcagatga ataccctgcg ggccgaggac accgccgtgt actactgcgc cagagacaga 360ggcatcggcg ccagacgggg cccttactac atggacgtgt ggggcaaggg caccaccgtg 420acagtgtctg gcggaggcgg aagtggcgga gggggatcag gcgggggagg cagcgatatt 480cagatgaccc agagccccag cagcctgagc gcctctgtgg gcgacagagt gaccatcacc 540tgtcgggcca gccagagcat cagctcctac ctgaactggt atcagcagaa gcccggcaag 600gcccccaagc tgctgatcta tgccgcctcc agtctgcaga gcggcgtgcc cagcagattt 660tctggcagcg gctccggcac cgacttcacc ctgacaatca gctccctgca gcccgaggac 720ttcgccacct actactgcca gcagagctac agcacccccc tgacattcgg cggaggcacc 780aaggtggaaa tcaagcacca ccaccatcac cactgatga 8198271PRTArtificial sequenceAducanumab scFv construct amino acid sequenceSIGNAL(1)..(20)IL2 secretion signalMISC_FEATURE(21)..(143)MISC_FEATURE(144)..(158)Gly-Ser LlinkerMISC_FEATURE(159)..(265)Light Variable regionMISC_FEATURE(266)..(271)6xHistag (Explasy) 8Met Tyr Lys Met Gln Leu Leu Ser Cys Ile Ala Leu Thr Leu Val Leu1 5 10 15Val Ala Asn Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val 20 25 30Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala 35 40 45Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu Glu Trp Val Ala Val Ile Trp Phe Asp Gly Thr Lys Lys Tyr Tyr65 70 75 80Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys 85 90 95Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Arg Asp Arg Gly Ile Gly Ala Arg Arg Gly Pro 115 120 125Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Gly 130 135 140Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile145 150 155 160Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg 165 170 175Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn 180 185 190Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala 195 200 205Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 210 215 220Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp225 230 235 240Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Leu Thr Phe 245 250 255Gly Gly Gly Thr Lys Val Glu Ile Lys His His His His His His 260 265 2709798DNAArtificial SequenceEngineered crenezumab scFv construct 9atgtacaaga tgcagctgct gagctgtatc gccctgaccc tggtgctggt ggccaactct 60gaagtgcagc tggtggaaag cggcggaggc ctggtgcagc ctggcggatc tctgagactg 120agctgtgccg ccagcggctt caccttcagc agctacggca tgagctgggt gcgccaggcc 180cctggaaaag gcctggaact ggtggcctcc atcaacagca atggcggcag cacctactac 240cccgacagcg tgaagggccg gttcaccatc tcccgggaca acgccaagaa cagcctgtac 300ctgcagatga actccctgcg ggccgaggac accgccgtgt actattgtgc cagcggcgac 360tattggggcc agggcacaac cgtgacagtg tctggcggag gcggatctgg gggcggagga 420tcaggcgggg gaggatctga tatcgtgatg acccagagcc ccctgagcct gcctgtgaca 480cctggcgaac ctgccagcat cagctgcaga tccagccaga gcctggtgta cagcaacggc 540gacacctacc tgcactggta tctgcagaag cccggccaga gccctcagct gctgatctac 600aaggtgtcca accggttcag cggcgtgccc gacagatttt ctggcagcgg ctccggcacc 660gacttcaccc tgaagatcag ccgggtggaa gccgaggacg tgggcgtgta ctactgcagc 720cagtccaccc acgtgccctg gacatttgga cagggcacca aggtggaaat caagcaccac 780caccatcacc actgatga 79810264PRTArtificial sequenceEngineered Crenezumab scFv amino acid sequenceSIGNAL(1)..(20)IL-2 secretion signalMISC_FEATURE(21)..(131)Heavy variable regionMISC_FEATURE(131)..(146)Gly-Ser LinkerMISC_FEATURE(147)..(258)Light Variable RegionMISC_FEATURE(259)..(264)6xHis tag 10Met Tyr Lys Met Gln Leu Leu Ser Cys Ile Ala Leu Thr Leu Val Leu1 5

10 15Val Ala Asn Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45Phe Ser Ser Tyr Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu Glu Leu Val Ala Ser Ile Asn Ser Asn Gly Gly Ser Thr Tyr Tyr65 70 75 80Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Thr Val 115 120 125Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140Gly Ser Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr145 150 155 160Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val 165 170 175Tyr Ser Asn Gly Asp Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly 180 185 190Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly 195 200 205Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser225 230 235 240Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 245 250 255Ile Lys His His His His His His 26011798DNAArtificial Sequenceengineered Solanezumab scFv construct nucleic acid sequence 11atgtacaaga tgcagctgct gagctgtatc gccctgaccc tggtgctggt ggccaactct 60gaagtgcagc tggtggaaag cggcggaggc ctggtgcagc ctggcggatc tctgagactg 120agctgtgccg ccagcggctt caccttcagc cggtacagca tgagctgggt gcgccaggcc 180cctggcaaag gactggaact ggtggcccag atcaacagcg tgggcaacag cacctactac 240cccgacaccg tgaagggccg gttcaccatc agcagagaca acgccaagaa caccctgtac 300ctgcagatga acagcctgcg ggccgaggac accgccgtgt actattgtgc cagcggcgac 360tattggggcc agggcacact cgtgacagtg tctggcggag gcggatctgg gggcggagga 420tcaggcgggg gaggatctga tgtcgtgatg acccagagcc ccctgagcct gcctgtgaca 480ctgggacagc ctgccagcat cagctgcaga agcagccaga gcctgatcta cagcgacggc 540aacgcctacc tgcactggtt tctgcagaaa cccggccagt cccccagact gctgatctat 600aaggtgtcca accggttcag cggcgtgccc gacagatttt ctggcagcgg ctccggcacc 660gacttcaccc tgaagatcag ccgggtggaa gccgaggacg tgggcgtgta ctactgcagc 720cagtccaccc acgtgccctg gacatttgga cagggcacca aggtggaaat caagcaccac 780caccatcacc actgatga 79812264PRTArtificial Sequenceengineered Solanezumab scFv construct amino acid sequenceSIGNAL(1)..(20)IL2 secretion signalMISC_FEATURE(21)..(131)Heavy chain variableMISC_FEATURE(132)..(146)MISC_FEATURE(147)..(258)Variable Light RegionMISC_FEATURE(259)..(264)6xHis tag 12Met Tyr Lys Met Gln Leu Leu Ser Cys Ile Ala Leu Thr Leu Val Leu1 5 10 15Val Ala Asn Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val 20 25 30Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr 35 40 45Phe Ser Arg Tyr Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly 50 55 60Leu Glu Leu Val Ala Gln Ile Asn Ser Val Gly Asn Ser Thr Tyr Tyr65 70 75 80Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala 100 105 110Val Tyr Tyr Cys Ala Ser Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val 115 120 125Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 130 135 140Gly Ser Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr145 150 155 160Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Ile 165 170 175Tyr Ser Asp Gly Asn Ala Tyr Leu His Trp Phe Leu Gln Lys Pro Gly 180 185 190Gln Ser Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly 195 200 205Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 210 215 220Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser225 230 235 240Gln Ser Thr His Val Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu 245 250 255Ile Lys His His His His His His 260133165DNAArtificial sequenceAAV vector genome CB7.CI.aducanumabscFvRBGrepeat_region(1)..(130)5" AAV2-ITRrepeat_region(198)..(579)CMV IE promoterpromoter(582)..(863)CB promoterTATA_signal(836)..(838)Intron(958)..(1930)chicken beta actin promotermisc_feature(1958)..(2001)misc_feature(2002)..(2370)aducanumab heavy variable regionmisc_feature(2371)..(2415)Gly-Ser Linkermisc_feature(2416)..(2736)aducanumab light chain variablemisc_feature(2737)..(2754)His TagpolyA_signal(2821)..(2947)Rabbit globulin polyarepeat_region(3036)..(3165)3' ITR 13ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta 420aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 1020gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg 1500cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920tttggcaaag aattcgccac catgtacaag atgcagctgc tgagctgtat cgccctgacc 1980ctggtgctgg tggccaactc tcaggtgcag ctggtggaat ctggcggcgg agtggtgcag 2040cctggcagaa gcctgagact gagctgtgcc gccagcggct tcgccttcag cagctacgga 2100atgcactggg tgcgccaggc ccctggcaaa ggactggaat gggtggccgt gatttggttc 2160gacggcacca agaagtacta caccgacagc gtgaagggcc ggttcaccat cagccgggac 2220aacagcaaga acaccctgta cctgcagatg aataccctgc gggccgagga caccgccgtg 2280tactactgcg ccagagacag aggcatcggc gccagacggg gcccttacta catggacgtg 2340tggggcaagg gcaccaccgt gacagtgtct ggcggaggcg gaagtggcgg agggggatca 2400ggcgggggag gcagcgatat tcagatgacc cagagcccca gcagcctgag cgcctctgtg 2460ggcgacagag tgaccatcac ctgtcgggcc agccagagca tcagctccta cctgaactgg 2520tatcagcaga agcccggcaa ggcccccaag ctgctgatct atgccgcctc cagtctgcag 2580agcggcgtgc ccagcagatt ttctggcagc ggctccggca ccgacttcac cctgacaatc 2640agctccctgc agcccgagga cttcgccacc tactactgcc agcagagcta cagcaccccc 2700ctgacattcg gcggaggcac caaggtggaa atcaagcacc accaccatca ccactgatga 2760ggtacctcta gagtcgaccc gggcggcctc gaggacgggg tgaactacgc ctgaggatcc 2820gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact 2880tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc 2940tcactcggaa gcaattcgtt gatctgaatt tcgaccaccc ataataccca ttaccctggt 3000agataagtag catggcgggt taatcattaa ctacaaggaa cccctagtga tggagttggc 3060cactccctct ctgcgcgctc gctcgctcac tgaggccggg cgaccaaagg tcgcccgacg 3120cccgggcttt gcccgggcgg cctcagtgag cgagcgagcg cgcag 3165143144DNAArtificial sequencepAAV.CB7.CI.crenezumabScFv.RBGrepeat_region(1)..(130)AAV2 - 5' ITRrepeat_region(198)..(579)CMV IE promoterpromoter(582)..(863)CB promoterTATA_signal(836)..(839)Intron(958)..(1930)misc_feature(1942)..(20- 01)IL-2 secretion signalmisc_feature(2001)..(2334)crenezumab heavy variable regionmisc_feature(2335)..(2379)Gly-Ser linkermisc_feature(2380)..(2715)crenezumab varalbe region lightmisc_feature(2716)..(2733)His tagpolyA_signal(2800)..(2926)rabbit globin polyArepeat_region(3015)..(3144)AAV2 - 3' ITR 14ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta 420aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 1020gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg 1500cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920tttggcaaag aattcgccac catgtacaag atgcagctgc tgagctgtat cgccctgacc 1980ctggtgctgg tggccaactc tgaagtgcag ctggtggaaa gcggcggagg cctggtgcag 2040cctggcggat ctctgagact gagctgtgcc gccagcggct tcaccttcag cagctacggc 2100atgagctggg tgcgccaggc ccctggaaaa ggcctggaac tggtggcctc catcaacagc 2160aatggcggca gcacctacta ccccgacagc gtgaagggcc ggttcaccat ctcccgggac 2220aacgccaaga acagcctgta cctgcagatg aactccctgc gggccgagga caccgccgtg 2280tactattgtg ccagcggcga ctattggggc cagggcacaa ccgtgacagt gtctggcgga 2340ggcggatctg ggggcggagg atcaggcggg ggaggatctg atatcgtgat gacccagagc 2400cccctgagcc tgcctgtgac acctggcgaa cctgccagca tcagctgcag atccagccag 2460agcctggtgt acagcaacgg cgacacctac ctgcactggt atctgcagaa gcccggccag 2520agccctcagc tgctgatcta caaggtgtcc aaccggttca gcggcgtgcc cgacagattt 2580tctggcagcg gctccggcac cgacttcacc ctgaagatca gccgggtgga agccgaggac 2640gtgggcgtgt actactgcag ccagtccacc cacgtgccct ggacatttgg acagggcacc 2700aaggtggaaa tcaagcacca ccaccatcac cactgatgag gtacctctag agtcgacccg 2760ggcggcctcg aggacggggt gaactacgcc tgaggatccg atctttttcc ctctgccaaa 2820aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt 2880attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag caattcgttg 2940atctgaattt cgaccaccca taatacccat taccctggta gataagtagc atggcgggtt 3000aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 3060ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 3120ctcagtgagc gagcgagcgc gcag 3144153144DNAArtificial SequencepAAV.CB7.CI.solanezumabscFv.RBGrepeat_region(1)..(130)AAV2 - 5' ITRrepeat_region(198)..(579)CMV IE promoterpromoter(582)..(863)CB promoterTATA_signal(836)..(839)Intron(958)..(1930)chicken beta-actin intronmisc_feature(1942)..(2001)IL-2 secretion signalmisc_feature(2002)..(2334)solanezumab heavy variable regionmisc_feature(2335)..(2379)Gly-Ser Linkermisc_feature(2380)..(2715)solanezumab light chain variable regionpolyA_signal(2800)..(2926)rabbit globin polyAmisc_feature(3015)..(3144)AAV2 - 3' ITR 15ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct tgtagttaat gattaacccg ccatgctact tatctaccag ggtaatgggg 180atcctctaga actatagcta gtcgacattg attattgact agttattaat agtaatcaat 240tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa 300tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt 360tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggact atttacggta 420aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 480caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc 540tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 600gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 660tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 720ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 780gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 840aaagcgaagc gcgcggcggg cggggagtcg ctgcgacgct gccttcgccc cgtgccccgc 900tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 960agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 1020gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1080gggagcggct cggggggtgc gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc 1140gcgctgcccg gcggctgtga gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt 1200gtgcgcgagg ggagcgcggc cgggggcggt gccccgcggt gcgggggggg ctgcgagggg 1260aacaaaggct gcgtgcgggg tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg 1320gtcgggctgc aaccccccct gcacccccct ccccgagttg ctgagcacgg cccggcttcg 1380ggtgcggggc tccgtacggg gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg 1440caggtggggg tgccgggcgg ggcggggccg cctcgggccg gggagggctc gggggagggg 1500cgcggcggcc cccggagcgc cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt 1560ttatggtaat cgtgcgagag ggcgcaggga cttcctttgt cccaaatctg tgcggagccg 1620aaatctggga ggcgccgccg caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc 1680ggcaggaagg aaatgggcgg ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc 1740cctctccagc ctcggggctg tccgcggggg gacggctgcc ttcggggggg acggggcagg 1800gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat 1860gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat 1920tttggcaaag aattcgccac catgtacaag atgcagctgc tgagctgtat cgccctgacc 1980ctggtgctgg tggccaactc tgaagtgcag ctggtggaaa gcggcggagg cctggtgcag 2040cctggcggat ctctgagact gagctgtgcc gccagcggct tcaccttcag ccggtacagc 2100atgagctggg tgcgccaggc ccctggcaaa ggactggaac tggtggccca gatcaacagc 2160gtgggcaaca gcacctacta ccccgacacc gtgaagggcc ggttcaccat cagcagagac 2220aacgccaaga acaccctgta cctgcagatg aacagcctgc gggccgagga caccgccgtg 2280tactattgtg ccagcggcga ctattggggc cagggcacac tcgtgacagt gtctggcgga 2340ggcggatctg ggggcggagg atcaggcggg ggaggatctg atgtcgtgat gacccagagc 2400cccctgagcc tgcctgtgac actgggacag cctgccagca tcagctgcag aagcagccag 2460agcctgatct acagcgacgg caacgcctac ctgcactggt ttctgcagaa acccggccag 2520tcccccagac tgctgatcta taaggtgtcc aaccggttca gcggcgtgcc cgacagattt 2580tctggcagcg gctccggcac cgacttcacc ctgaagatca gccgggtgga agccgaggac 2640gtgggcgtgt actactgcag ccagtccacc cacgtgccct ggacatttgg acagggcacc 2700aaggtggaaa tcaagcacca ccaccatcac cactgatgag gtacctctag agtcgacccg 2760ggcggcctcg aggacggggt

gaactacgcc tgaggatccg atctttttcc ctctgccaaa 2820aattatgggg acatcatgaa gccccttgag catctgactt ctggctaata aaggaaattt 2880attttcattg caatagtgtg ttggaatttt ttgtgtctct cactcggaag caattcgttg 2940atctgaattt cgaccaccca taatacccat taccctggta gataagtagc atggcgggtt 3000aatcattaac tacaaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg 3060ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc 3120ctcagtgagc gagcgagcgc gcag 3144

Claims

1. A composition formulated for intrathecal delivery comprising at least one recombinant adeno-associated virus (rAAV) for treatment of a neurodegenerative disorder selected from one or more of Parkinson's disease, amyotropic lateral sclerosis (ALS), multiple sclerosis, Alzheimer's Disease, Huntington disease, traumatic brain injury, spinal cord injury, migraine, stroke, and/or an infectious disease, wherein said at least one rAAV comprises an AAV capsid which targets a cell of the central nervous system, the capsid having packaged therein sequences encoding an immunoglobulin construct operably linked to expression control sequences therefor, the composition further comprising a pharmaceutically acceptable carrier and/or excipient.

2. The composition according to claim 1, wherein the immunoglobulin construct is selected from a single chain variable fragment antibody (scFv), an Fv, Fab, F(ab).sub.2, F(ab).sub.3, Fab', Fab'-SH, F(ab').sub.2, an immunoadhesin, a monoclonal antibody, a heavy chain camelid or shark immunoglobulins.

3. The composition according to claim 1, wherein the disorder is Alzheimer's Disease and the composition comprises at least one rAAV stock expressing an immunoglobulin specific for a A.beta., beta secretase, and/or tau protein.

4. The composition according to claim 3, wherein the immunoglobulin is a scFv.

5. The composition according to claim 4, wherein the scFv has the amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO:12.

6. The composition according to claim 1, wherein the composition comprises rAAV stock comprising at least two different antibody expression cassettes.

7. The composition according to claim 1, wherein the composition comprises a AAV stock comprising a single immunoglobulin expression cassette.

8. The composition according to claim 1, wherein the composition is delivered intrathecally and treats the condition both in the central nervous system and systemically.

10. The composition according to claim 1, wherein the immunoglobulin construct comprises an immunoglobulin modified to have decreased or no measurable affinity for neonatal Fc receptor (FcRn).

11. A composition useful for treatment of Alzheimer's Disease comprising at least one AAV vector stock comprising an AAV capsid which targets a cell of the central nervous system, the capsid having packaged therein at least one AAV inverted terminal repeat sequence and sequences encoding an immunoglobulin construct operably linked to expression control sequences therefor, wherein the at least one vector stock expresses an immunoglobulin specific for a A.beta., beta secretase, and/or the tau protein, and the composition further comprises a pharmaceutically acceptable carrier and/or excipient.

12. The composition according to claim 11, wherein the immunoglobulin is a scFv.

13. The composition according to claim 12, wherein the scFv has the amino acid sequence selected from SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO:12.

14. A method for treatment of Alzheimer's Disease, said method comprising delivering intrathecally a composition according to claim 1, in which the composition comprises at least one vector stock expressing an immunoglobulin specific for a A.beta., beta secretase, and/or the tau protein, to subject in need thereof.

15. A method for treatment of ALS, said method comprising delivering intrathecally a composition according to claim 1 which comprises at least one rAAV expressing an immunoglobulin specific for a Derlin-1-binding region, an antibody construct against neurite outgrowth inhibitor, or ALS enzyme superoxide dismutase 1 (SOD1) and variants thereof, provided the anti-SOD1 antibody is other than an ScFv fragment.

16. A method for treatment of Parkinson's Disease or related synucleinopathies, said method comprising delivering intrathecally a composition according to claim 1 which comprises at least one rAAV stock encoding one or more leucine-rich repeat kinase 2 antibody, dardarin (LRRK2) antibody, alpha-synuclein antibody, or DJ-1 (PARK7) antibody.

17. A method for treatment of multiple sclerosis, said method comprising delivery intrathecally of a composition according to claim 1 which comprises at least one rAAV stock encoding an immunoglobulin directed against one or more of an a4-integrin, LINGO1, CD20, CD25, IL12, p40+IL23p40, LINGO-1, CD40, and rHIgM22, CD52, IL17, CD19, or SEMA4D.

18. A method for treatment of infectious disease of the central nervous system, said method comprising delivering a composition according to claim 1 intrathecally which comprises at least one vector stock encoding an immunoglobulin directed against one of Mycobacterium tuberculosis, Neisseria meningitides, Streptococcus pneumonia, Listeria monocytogens, Borrelia burdorferia, human immunodeficiency virus, a herpes family viruses, varicella zoster virus, Epstein-Barr virus (EBV), cytomegalovirus, japanese encephalitis, european encephalitis, and/or JC virus.

19. A method for treatment of prion related diseases, said method comprising delivering a composition according to claim 1 intrathecally, which composition comprises at least one vector stock encodes an immunoglobulin directed one or more of major prion protein, or PrP.sup.Sc.

20. The method according to claim 19, wherein said composition is delivered in the absence of chemical or physical disruption of the blood brain barrier.