CD20 antibody AAV vectors and methods of use thereof

Abstract

The disclosure is directed to adeno-associated viral (AAV) vector comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain, and uses of same for CD20 positive cancers of the central nervous system.

Description

CD20 ANTIBODY AAV VECTORS AND METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to, and benefit of, U.S. Provisional Application No. 63 / 587,518, filed October 3, 2023, the contents of which are incorporated by reference in their entirety herein. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (BRAV- 006_001WO_SEQLIST_st26.xml; Size: 217,088 bytes; and Date of Creation: September 27, 2024) are herein incorporated by reference in their entirety. BACKGROUND

[0003] Many therapeutic antibodies, when administered systemically, do not cross the blood brain barrier to an extent that would allow them to be used to effectively treat central nervous system (CNS) cancers. However, there are a number of CNS cancers that could benefit from treatment with antibody-based immunotherapy. Two examples of such cancers are primary CNS lymphoma (PCNSL), a rare form of non-Hodgkin lymphoma that remains isolated in the CNS, and secondary CNS lymphoma. PCNSL is found in the brain in approximately 80% of cases. Patients usually present with severe neurological symptoms, and diagnosis is made based on contrast-enhanced MRI imaging combined with corresponding patient evaluation. Prognosis remains poor, with estimated overall five-year survival rates ranging from 30-40% with current treatment options. The majority of patients experience relapse within two years, and overall survival after relapse is typically less than year. Patients over the age of 60 have an even poorer prognosis. There are approximately 1,500 new cases of PCNSL diagnosed in the U.S. each year in immunocompetent patients. However, there is no uniform consensus on the optimal treatment regimen for patients with PCNSL. Treatment is largely based on individual patient’s disease characteristics, age and / or comorbidities, and the personal preferences of the treating physicians. However, those therapies that are generally used, such as high -dose methotrexate, have high toxicity, limited patient eligibility, and poor efficacy. There thus is an unmet need for additional treatments for CNS cancers such as PCNSL and secondary CNS lymphoma. The current disclosure provides AAV vectors encoding CD20 antibodies, compositions comprising these AAV vectors, and use of these vectors for the treatment of CNS cancers such as PCNSL and secondary CNS lymphoma.SUMMARY

[0004] The disclosure provides adeno-associated viral (AAV) vectors comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain.

[0005] In some embodiments of the AAV vectors of the disclosure, the AAV vector comprises one or more modifications to the capsid which increase the efficiency with which the AAV vector crosses the blood brain barrier in a subject relative to an AAV vector comprising an unmodified capsid. In some embodiments, the one or more modifications comprises a targeting peptide in the capsid, for example TVSALFK (SEQ ID NO: 96) or TVSALK (SEQ ID NO: 92).

[0006] The disclosure provides pharmaceutical compositions comprising the AAV vectors as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0007] The disclosure provides methods of treating a cancer in a subject, comprising administering a therapeutically effective amount of the AAV vectors or the pharmaceutical compositions of the disclosure.

[0008] The disclosure provides AAV vectors comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain, and pharmaceutical compositions comprising same, for use in treating a cancer in a subject in need thereof.

[0009] The disclosure provides AAV vectors comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain, for use in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof.

[0010] In some embodiments, the cancer comprises a central nervous system cancer. In some embodiments, the cancer comprises primary central nervous system lymphoma (PCNSL) or secondary central nervous system lymphoma.

[0011] The disclosure provides polynucleotides encoding the AAV vectors, and components of the AAV vectors, described herein.

[0012] The disclosure provides kits comprising the AAV vectors described herein, and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG.1 Is a schematic of an exemplary AAV vector for expression of the CD20 antibody rituximab. Abbreviations: SP1: Signal Peptide 1 (for Heavy Chain); SP2: Signal Peptide 2 (for Light Chain); ITR: inverted terminal repeat.

[0014] FIG.2 is a table showing candidate AAV vectors encoding rituximab. CAG: CMV early enhancer / chicken beta actin hybrid promoter; hSyn: Homo sapiens synapsin 1; GFAP: Homo sapiens glial fibrillary acidic protein; CBA: chicken beta actin promoter; HGH: human growth hormone; IgKVIII: Ig kappa chain V-III region; H7: histocompatibility 7; GRN: granulin; HGH1: IL2: interleukin 2.

[0015] FIG.3 is a bar plot showing rituximab in 293T cell media following transient transfection of 293T cells with the expression vectors listed in FIG.2.

[0016] FIG.4 is a Western blot showing that CD20 antibody expressed by 293T cells transfected with the vectors shown in FIG.1 is processed into heavy and light chains.

[0017] FIG.5A is a plot showing rituximab levels in plasma of mice treated with a single dose of a rituximab AAV expression vector with the elements shown in FIG.2.

[0018] FIG.5B is a plot showing rituximab levels in the brains of mice treated with a single dose of a rituximab AAV expression vector with the elements shown in FIG.2.

[0019] FIG.5C is a plot showing rituximab levels in the skeletal muscle of mice treated with a single dose of a rituximab AAV expression vector with the elements shown in FIG.2.

[0020] FIG.5D is a plot showing rituximab levels in the liver of mice treated with a single dose of a rituximab AAV expression vector with the elements shown in FIG.2. DETAILED DESCRIPTION

[0021] Approximately 84% of PCNSLs in the brain are diffuse large B-cell lymphomas (DLBCL) which are CD20-positive. Anti-CD20 antibodies, such as rituximab, have been found to be efficacious in treating B-cell lymphomas located outside the central nervous system. Rituximab is also known to be safe, as chronic rituximab treatment has routinely been used for over 10 years in rheumatoid arthritis (RA) patients. As one example, R-CHOP (rituximab in combination with cyclophosphamide, doxorubicin, vincristine, and prednisone), has been found to cure diffuse large B-cell lymphomas (DLBCL) outside the CNS in approximately 70% of patients. However, the ability of antibodies such as rituximab to cross the blood brain barrier when administered systemically, and thus reach CNS cancers, is poor. While direct intraventricular (IVC) dosing is one possibility, this approach is generally not sustainable due to the poor PK and short half-life of antibodies in cerebrospinal fluid, which requires multiple administrations, and the invasiveness of the procedure. By using an AAVcapable of crossing the blood brain barrier to encode CD20 antibodies, the disclosure provides compositions and methods for treating CD20+ CNS cancers. Definitions

[0022] As used herein, the term “adeno-associated virus” or “AAV” refers to members of the dependoparvovirus genus, and includes any vector, sequence, gene, protein, or component derived therefrom. The AAV genome is built of single-stranded deoxyribonucleic acid (ssDNA). At each end of the DNA strand is an inverted terminal repeat (ITR). Between the ITRs are two open reading frames (ORFs): Rep and Cap. The Rep ORF is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle. The Cap ORF contains overlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. The term AAV includes without limitation, type 1, AAV type 2, AAV type 3 (including types 3 A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type rh.10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV and any other AAV now known or later discovered.

[0023] The genomic sequences of various AAV and autonomous parvoviruses, as well as the sequences of the ITRs, rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as the GENBANK database. The term AAV includes, without limitation, AAV generated through any methods known in the art. For example, AAV includes recombinant AAV (rAAV), which refers to AAV that are not naturally occurring, and can be generated using recombinant techniques. Recombinant AAV can refer to AAV in which part of the AAV, such as the capsid or the AAV genome, has been modified using recombinant DNA techniques known in the art. As a further example, recombinant AAV can also refer to an AAV in which part of the AAV has been derived a first source (e.g., AAV2 ITRs), while the rest of the AAV has been derived a second source (e.g., AAV9 capsid).

[0024] As used herein, a “vector” is any molecule or moiety which transports, transduces, or otherwise acts as a carrier of a heterologous molecule such as the nucleic acids described herein. This includes the AAV vectors described herein.

[0025] As used herein, the term “AAV vector” refers to a viral genome packaged in an AAV capsid. In some cases, the viral genome of the AAV vector comprises minimal AAV sequences to avoid replication (e.g., the Rep and Cap are removed, leaving the invertedterminal repeat sequences) and a transgene, such as a transgene encoding a CD20 antigen binding domain.

[0026] As used herein, “viral genome” refers to the nucleic acid sequence(s) encapsulated in an AAV particle. Exemplary viral genomes comprise at least one transgene encoding a polypeptide, e.g., an antibody, antibody-based composition or fragments thereof, flanked by AAV ITRs.

[0027] As used herein, the term “antigen binding domain” refers to a protein, or a domain of a protein, that is capable of binding to an antigen. Antigen binding domains can be, without limitation, antibodies or antibody fragments.

[0028] As used herein, an “antigen” is an entity which induces or evokes an immune response in an organism. An immune response is characterized by the reaction of the cells, tissues and / or organs of an organism to the presence of a foreign entity. Such an immune response typically leads to the production by the organism of one or more antibodies against the foreign entity, e.g., antigen or a portion of the antigen. As used herein, “antigens” also refer to binding partners for specific antibodies or binding agents in a display library.

[0029] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.

[0030] As used herein, the term “nucleic acid,” “polynucleotide” or similar refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double- stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res.19:5081, 1991; Ohtsuka et al., Biol. Chem.260:2605-2608, 1985; and Cassol et al, 1992; Rossolini et al, Mol. Cell. Probes 8:91- 98, 1994). For arginine and leucine, modifications at the second base can also beconservative. The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.

[0031] Polynucleotides used herein can be composed of any polyribonucleotide or polydeoxribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. For example, polynucleotides can be composed of single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that can be single- stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the polynucleotide can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "polynucleotide" embraces chemically, enzymatically, or metabolically modified forms.

[0032] A polypeptide or polynucleotide sequence “derived from” a designated polypeptide or polynucleotide refers to the origin of the polypeptide or polynucleotide. Preferably, the polypeptide or polynucleotide sequence which is derived from a particular sequence has an acid sequence that is essentially identical to, or highly similar to, that particular sequence or a portion thereof, and is identifiable to one of ordinary skill in the art as having its origin in that particular sequence. Polypeptides or polynucleotides derived from another peptide or polynucleotide may have one or more mutations relative to the starting sequence, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. Polypeptides or polynucleotides derived from another peptide or polynucleotide may be incorporated into fusion proteins or sequences encoding fusion proteins. Alternatively, a polypeptide or polynucleotide which is identical to a portion of an original from which it is derived, but is shorter than the original, can be said to be derived from the original polypeptide or polynucleotide.

[0033] It will also be understood by one of ordinary skill in the art that the polypeptides and polynucleotides suitable for use in the compositions and methods disclosed herein may be altered such that they vary in sequence from the sequences from which they were derived, while retaining the desirable activity of the original sequences. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential”amino acid residues may be made. Mutations may be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis.

[0034] In general, “homology” or “identical”, percent “identity” or “similarity”, when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, e.g., at least 60% identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein). Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. The alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Current Protocols in Molecular Biology (Ausubel et al., eds.1987) Supplement 30, section 7.7.18, Table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, using default parameters. Details of these programs can be found at the following Internet address: ncbi.nlm.nih.gov / cgi-bin / BLAST. The terms “homology” or “identical”, percent “identity” or “similarity” also refer to, or can be applied to, the complement of a test sequence. The terms also include sequences that have deletions and / or additions, as well as those that have substitutions. As described herein, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0035] The polypeptides suitable for use in the compositions and methods disclosed herein may comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a binding polypeptide is preferably replaced with another amino acid residue from the same side chain family. A “non-conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue that does not have a similar side chain.

[0036] As used herein, the term “protease” refers to naturally-occurring polypeptides (or polypeptides derived from naturally-occurring polypeptides which retain protease activity) that cleave peptide bonds of proteins or peptides. “Protease” encompasses mature forms of proteases, as well as the pro- and pre-forms of the protease. Proteases that cleave proteins are also referred to as “proteinases.” Exemplary proteases include, but are not limited to, endoproteases such as furin. Further exemplary proteases include matrix metalloproteases.

[0037] As used here, the term “protease sensitive domain” refers to a region in a polypeptide that increases susceptibility of the polypeptide to cleavage by a protease (e.g. a proteinase or peptidase). As a non-limiting example, the furin protease is known to cleave the sequence RKRR (SEQ ID NO: 161) and similar sequences.

[0038] As used herein, the term “self-cleaving peptide” refers to a peptide which can induce ribosomal skipping during translation, producing two separate polypeptides from the same mRNA transcript. Exemplary, but non-limiting, self-cleaving peptides include T2A, E2A, F2A and P2A peptides.

[0039] As used herein, the term “internal ribosome entry site (IRES)” refers to a sequence used to express two polypeptides from a single mRNA transcript by the introduction of a second translational start site on the mRNA.

[0040] The term “promoter” refers to a DNA sequence that contains a transcription start site and additional sequences that facilitate polymerase binding and transcription. Exemplary eukaryotic promoters include a TATA box, and / or B recognition element (BRE) and act to promote the transcription associated transcribable polynucleotide sequence, such as a sequence encoding an antibody. A promoter can be synthetically produced or can be derived from a known or naturally occurring promoter . Promoters can be ubiquitous, i.e. driving expression in all cells in the body, or can be tissue-specific, i.e. driving expression only in a specific tissue or tissues.

[0041] As used herein, “systemic circulation” refers to the entirety of components carried along with oxygenated blood by the cardiovascular system as it carries oxygenated bloodaway from the heart, to the body, and returns deoxygenated blood back to the heart. Components of the systemic circulation include, but are not limited to, serum, blood plasma, blood cells, red blood cells, white blood cells, antibodies, proteins, nucleic acids, and immune cells. “Systemic administration” encompasses routes of administration that put the agent administered into systemic circulation, such as intravenous injection or infusion.

[0042] As used herein “intrathecal administration” refers to a route of administration that delivers an agent into the spinal canal or subarachnoid space, and is often used to bypass the blood brain barrier (BBB). Exemplary intrathecal routes include intracerebroventricular (ICV) injection, intracisterna magna (ICM) injection or injection into the subarachnoid space of the spinal cord (IT) injection. In ICV injection, agents such as the AAV vectors described herein are injected into the cerebral ventricles so that they reach the cerebrospinal fluid (CSF). For intra-cisterna magna (ICM) injection, the agent injected is in the cerebrospinal fluid (CSF)-filled subarachnoid space between the cerebellum and the dorsal side of the medulla oblongata.

[0043] As used herein “Primary CNS lymphoma (PCNSL)” refers to a type of cancer originating from immune cells known as lymphocytes (lymphoma) that develops in the brain and / or spinal cord.

[0044] “Secondary central nervous system lymphoma (SCNSL)” refers to a rare cancer, which occurs when there is secondary central nervous system (CNS) involvement in patients with systemic lymphoma.

[0045] As used herein, “treating” or “treat” describes the management and care of a subject for the purpose of combating a disease, condition, or disorder, such as the cancers described herein. “Treat” or “treating” as used herein includes the administration of AAV vectors of the disclosure to a subject with cancer to alleviate the signs or symptoms of cancer, or to eliminate the cancer.

[0046] The term “alleviate” is meant to describe a process by which the severity of a sign or symptom of cancer is decreased. Importantly, a sign or symptom can be alleviated without being eliminated. In a preferred embodiment, the administration of AAV vectors of the disclosure leads to the elimination of a sign or symptom, however, elimination is not required. Effective dosages are expected to decrease the severity of a sign or symptom of the cancer. For instance, a sign or symptom of a disorder such as cancer, which can occur in multiple locations, is alleviated if the severity of the cancer is decreased within at least one of multiple locations.

[0047] As used herein, the term “severity” is meant to describe the potential of cancer to transform from a precancerous, or benign, state into a malignant state. Alternatively, or in addition, severity is meant to describe a cancer stage, for example, according to the TNM system (accepted by the International Union Against Cancer (UICC) and the American Joint Committee on Cancer (AJCC)) or by other art-recognized methods. Cancer stage refers to the extent or severity of the cancer, based on factors such as the location of the primary tumor, tumor size, number of tumors, and lymph node involvement (spread of cancer into lymph nodes). Alternatively, or in addition, severity is meant to describe the tumor grade by art- recognized methods (see, National Cancer Institute, www.cancer.gov). Tumor grade is a system used to classify cancer cells in terms of how abnormal they look under a microscope and how quickly the tumor is likely to grow and spread. Many factors are considered when determining tumor grade, including the structure and growth pattern of the cells. The specific factors used to determine tumor grade vary with each type of cancer. Severity also describes a histologic grade, also called differentiation, which refers to how much the tumor cells resemble normal cells of the same tissue type (see, National Cancer Institute, www.cancer.gov). Furthermore, severity describes a nuclear grade, which refers to the size and shape of the nucleus in tumor cells and the percentage of tumor cells that are dividing (see, National Cancer Institute, www.cancer.gov).

[0048] As used herein, the term “refractory” describes a cancer that does not respond to an attempted form of treatment. Refractory cancers can also be termed resistant cancers.

[0049] As used herein the term “symptom” is defined as an indication of disease, illness, injury, or that something is not right in the body. Symptoms are felt or noticed by the individual experiencing the symptom, but may not easily be noticed by others. Others are defined as non-health-care professionals.

[0050] As used herein the term “sign” is also defined as an indication that something is not right in the body. But signs are defined as things that can be seen by a doctor, nurse, or other health care professional.

[0051] As used herein, the term “therapeutically effective amount” means an amount of viral vector or agent to be delivered by the viral vector (e.g., an antibody) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, improve symptoms of, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition.

[0052] As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term“therapeutically effective dose” is defined as an amount sufficient to at least partially arrest the disease and its complications in a subject already suffering from the disease. The term “therapeutically effective dose” can also refer to an amount sufficient to cure the disease in a subject already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the subject’s own immune system.

[0053] As used herein, “monotherapy” refers to the administration of a single active or therapeutic agent to a subject in need thereof. Preferably, monotherapy will involve administration of a therapeutically effective amount of an active agent, for example, cancer monotherapy with one of the AAV vectors of the present disclosure. Monotherapy may be contrasted with combination therapy, in which a combination of multiple active agents is administered, preferably with each component of the combination present in a therapeutically effective amount.

[0054] A “subject” is a mammal. Mammals include, but are not limited to, domesticated animals, non-human primates, humans, dogs, rabbits, mice, rats and other rodents. In some cases a subject is a human suffering from a disease or disorder, such as a cancer as described herein.

[0055] As used herein, the term “operably linked” refers to a first molecule being joined to a second molecule such that the first molecule is positioned in a way that it affects the function of the second molecule. Two molecules may or may not be part of a continuous single molecule and may be adjacent or non-adjacent. For example, a promoter is operably linked to a transcribable polynucleotide if the promoter regulates transcription of the transcribable polynucleotide molecule in a cell. As a further example, a signal peptide is operably linked to a protein if it has ability to affect localization of the protein, even if it is not directly linked to the protein (e.g., it is indirectly linked via intervening sequences).

[0056] Where a range of values is provided, it is understood that endpoints are included and that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included.

[0057] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0058] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0059] It will be appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. In other cases, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. It is intended that all combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0060] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. CD20 Antigen Binding Domains

[0061] The disclosure provides AAV vectors comprising sequences encoding CD20 antigen binding domains, compositions comprising same, and uses of same in the treatment of cancer.

[0062] Cluster of differentiation 20 (CD20), also known as Membrane-spanning 4-domains subfamily A member 1 (MS4A1), is a transmembrane protein that is expressed on the surface of human B-lymphocytes throughout maturation, with exception that expression is lost in terminally differentiated plasma cells. An exemplary human CD20 sequence comprises: 1 MDFSRRSFHR SLSSSLQAPV VSTVGMQRLG TTPSVYGGAG GRGIRISNSR HTVNYGSDLT 61 GGGDLFVGNE KMAMQNLNDR LASYLEKVRT LEQSNSKLEV QIKQWYETNA PRAGRDYSAY 121 YRQIEELRSQ IKDAQLQNAR CVLQIDNAKL AAEDFRLKYE TERGIRLTVE ADLQGLNKVF 181 DDLTLHKTDL EIQIEELNKD LALLKKEHQE EVDGLHKHLG NTVNVEVDAA PGLNLGVIMN 241 EMRQKYEVMA QKNLQEAKEQ FERQTAVLQQ QVTVNTEELK GTEVQLTELR RTSQSLEIEL 301 QSHLSMKESL EHTLEETKAR YSSQLANLQS LLSSLEAQLM QIRSNMERQN NEYHILLDIK 361 TRLEQEIATY RRLLEGEDVK TTEYQLSTLE ERDIKKTRKI KTVVQEVVDG KVVSSEVKEV421 EENI (SEQ ID NO: 50).

[0063] Accordingly, the disclosure provides AAV vectors comprising sequences encoding antigen binding domains that bind to a protein comprising a sequence of SEQ ID NO: 50, or a fragment thereof. In some embodiments, CD20 or a fragment thereof expressed by a cancer cell, for example a lymphoma cell. In some embodiments, the lymphoma cell is a primary central nervous system lymphoma (PCNSL) or secondary central nervous system lymphoma cell.

[0064] In some embodiments, the antigen binding domain comprises an antibody, an scFv, an Fv, a Fab fragment, a Fab’ fragment, a F(abƍ)2 fragment, a diabody, a single-chain antibody, or a single domain antibody.

[0065] In some embodiments, the antigen binding domain comprises an antibody. As used herein, the term “antibody refers to a whole (or full length) antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The IgG isotype can be further subdivided into four subclasses: IgG1, IgG2, IgG3 and IgG4. The term antibody encompasses all isotypes and subclasses. The term “antibody” also includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody, all of which are envisaged as within the scope of the disclosure. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.

[0066] Full length antibodies comprise two heavy chains and two light chains connected by disulfide bonds. Each heavy chain is paired with one of the light chains to which it is directly bound via a disulfide bond. Each heavy chain comprises a constant region (which varies depending on the isotype of the antibody) and a variable region. The variable region comprises three hypervariable regions (or complementarity determining regions (CDRs)) which are designated CDR-H1, CDR-H2 and CDR-H3 and which are supported within framework regions. Each light chain comprises a constant region and a variable region, with the variable region comprising three hypervariable regions (designated CDR-L1, CDR-L2 and CDR-L3) supported by framework regions in an analogous manner to the variable region of the heavy chain.

[0067] The hypervariable regions of each pair of heavy and light chains mutually cooperate to provide an antigen binding site that is capable of binding a target antigen. The binding specificity of a pair of heavy and light chains is defined by the sequences of CDR1, CDR2 and CDR3 of the heavy and light chains. Thus once a set of CDR sequences (i.e. the sequence of CDR1, CDR2 and CDR3 for the heavy and light chains) is determined which gives rise to a particular binding specificity, the set of CDR sequences can, in principle, be inserted into the appropriate positions within any other antibody framework regions linked with any antibody constant regions in order to provide a different antibody with the same antigen binding specificity.

[0068] Also included in the disclosure are antibody fragments capable of binding to CD20 or a portion thereof. The terms “antibody fragment,” “antigen-binding fragment,” “antigen binding portion,” “antigen binding domain” and similar terms refer to a fragment of an antibody that retains the ability to bind to a target antigen. Such fragments include, e.g., a single-chain antibody, variable fragment (Fv), a single chain Fv fragment (scFv), a bispecific Fab dimer (Fab2), a trispecific Fab trimer (Fab3), a bis-scFv, (scFv)2, a Camel Ig, a VHH, and Ig NAR, a disulfide stabilized Fv protein (dsFv), a single-domain antibody (sdAb), a diabody, an Fd fragment, a Fab fragment, a Fab’ fragment, or an F(ab’)2 fragment. An scFv is a single polypeptide chain that includes both the heavy and light chain variable regions of the antibody from which the scFv is derived. In addition, intrabodies, minibodies, triabodies, tetrabodies, and diabodies are also included in the definition of antibody and are compatible for use in the methods described herein. See, e.g., Todorovska et al., (2001) J. Immunol. Methods 248(l):47-66; Hudson and Kortt, (1999) J. Immunol. Methods 231(1): 177-189; Poljak, (1994) Structure 2(12): 1121-1123; Rondon and Marasco, (1997) Annu. Rev. Microbiol.51:257-283, the disclosures of each of which are incorporated herein by reference in their entirety.

[0069] The antibodies encoded by the AAV vectors of the disclosure may be monospecific for CD20, or bispecific. A “bispecific” or “bifunctional” antibody refers to an artificial hybrid antibody having two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai & Lachmann, (1990) Clin. Exp. Immunol.79:315-321; Kostelny et al., (1992) J. Immunol.148: 1547-1553.

[0070] The CD20 antibodies of the disclosure may include one or more mutations in the constant region that affect binding to the Fc receptor, and which modulate the immune response to the antibody. As used herein, the term “Fc receptor” refers to a polypeptide foundon the surface of immune effector cells, which is bound by the Fc region of an antibody. In some embodiments, the Fc receptor is an FcȖ receptor. There are three subclasses of FcJ receptors, FcȖRI (CD64), FcyRII (CD32) and FycRIII (CD16). All four IgG isotypes (IgGl, IgG2, IgG3 and IgG4) bind and activate Fc receptors FcȖRI, FcȖRIIA and FcȖRIIIA. FcȖRIIB is an inhibitory receptor, and therefore antibody binding to this receptor does not activate complement and cellular responses. FcȖRI is a high affinity receptor that binds to IgG in monomeric form, whereas FcȖRIIA and FcȖRIIA are low affinity receptors that bind IgG only in multimeric form and have slightly lower affinity. The binding of an antibody to an Fc receptor is governed by specific residues or domains within the Fc regions. Binding also depends on residues located within the hinge region and within the CH2 portion of the antibody. In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is dependent on binding of the Fc region to the Fc receptor (e.g., FcȖR). In some embodiments, the agonistic and / or therapeutic activity of the antibodies described herein is enhanced by binding of the Fc region to the Fc receptor (e.g., FcȖR).

[0071] In some embodiments, the CD20 antigen binding domain comprises an antibody or an antibody fragment. In some embodiments, the antibody or antibody fragment comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises a sequence of CDR-H1, CDR-H2 or CDR-H3 described in Table 1 below, or a sequence having 1, 2 or 3 substitutions, insertions or deletions relative thereto, and the light chain variable region comprises a sequence of CDR- L1, CDR-L2 or CDR-L3 as described in Table 1 below, or a sequence having 1, 2 or 3 substitutions, insertions or deletions relative thereto. In some embodiments, the heavy chain variable region comprises sequences of CDR-H1, CDR-H2 and CDR-H3 described in Table 1 below, and the light chain variable region comprises sequences of CDR-L1, CDR-L2 and CDR-L3 as described in Table 1 below.

[0072] CD20 antigen binding domains can be isolated or derived from any CD20 antibodies known in the art. For example, CD20 antigen binding domains can be isolated or derived from rituximab, ofatumumab, obinutuzumab, ibritumomab, ocrelizumab or ublituximab. In some embodiments, the CD20 antigen binding domain is isolated or derived from rituximab, ofatumumab or obinutuzumab. In some embodiments, the CD20 antigen binding domain is isolated or derived from rituximab.

[0073] In some embodiments, the AAV vector comprises a sequence encoding an antibody, and the antibody comprises rituximab, ofatumumab, obinutuzumab, ibritumomab,ocrelizumab or ublituximab. In some embodiments, the antibody comprises rituximab, ofatumumab or Obinutuzumab. In some embodiments, the antibody comprises rituximab.

[0074] In some embodiments, the heavy chain variable region comprises a complementarity determining region 1 (CDR-H1) comprising a sequence of SYNMH (SEQ ID NO: 2), a CDR-H2 comprising a sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 3), and a CDR-H3 comprising a sequence of STYYGGDWYFNV (SEQ ID NO: 4), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the light chain variable region comprises a CDR-L1 comprising a sequence of RASSSVSYIH (SEQ ID NO: 5), a CDR-L2 comprising a sequence of ATSNLAS (SEQ ID NO: 6) and a CDR-L3 comprising a sequence of QQWTSNPPT (SEQ ID NO: 7), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the heavy chain variable region comprises a CDR-H1 comprising a sequence of SYNMH (SEQ ID NO: 2), a CDR-H2 comprising a sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 3), and a CDR-H3 comprising a sequence of STYYGGDWYFNV (SEQ ID NO: 4); and the light chain variable region comprises a CDR-L1 comprising a sequence of RASSSVSYIH (SEQ ID NO: 5), a CDR-L2 comprising a sequence of ATSNLAS (SEQ ID NO: 6) and a CDR-L3 comprising a sequence of QQWTSNPPT (SEQ ID NO: 7).

[0075] In some embodiments, the heavy chain variable region comprises a CDR-H1 comprising a sequence of DYAMH (SEQ ID NO: 8), a CDR-H2 comprising a sequence of TISWNSGSIGYADSVKG (SEQ ID NO: 9), and a CDR-H3 comprising a sequence of DIQYGNYYYGMDV (SEQ ID NO: 10), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the light chain variable region comprises a CDR-L1 comprising a sequence of RASQSVSSYLA (SEQ ID NO: 11), a CDR-L2 comprising a sequence of DASNRAT (SEQ ID NO: 12) and a CDR-L3 comprising a sequence of QQRSNWPIT (SEQ ID NO: 13), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the heavy chain variable region comprises a CDR-H1 comprising a sequence of DYAMH (SEQ ID NO: 8), a CDR-H2 comprising a sequence of TISWNSGSIGYADSVKG (SEQ ID NO: 9), and a CDR-H3 comprising a sequence of DIQYGNYYYGMDV (SEQ ID NO: 10); and the light chain variable region comprises a CDR-L1 comprising a sequence of RASQSVSSYLA (SEQ ID NO: 11), a CDR-L2 comprising a sequence of DASNRAT (SEQ ID NO: 12) and a CDR-L3 comprising a sequence of QQRSNWPIT (SEQ ID NO: 13).

[0076] In some embodiments, the heavy chain variable region comprises a CDR-H1 comprising a sequence of YSWIN (SEQ ID NO: 14), a CDR-H2 comprising a sequence of RIFPGDGDTDYNGKFKG (SEQ ID NO: 15), and a CDR-H3 comprising a sequence of NVFDGYWLVY (SEQ ID NO: 16), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the light chain variable region comprises a CDR-L1 comprising a sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 17), a CDR-L2 comprising a sequence of QMSNLVS (SEQ ID NO: 18) and a CDR-L3 comprising a sequence of AQNLELPYT (SEQ ID NO: 19), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences. In some embodiments, the heavy chain variable region comprises a CDR-H1 comprising a sequence of YSWIN (SEQ ID NO: 14), a CDR-H2 comprising a sequence of RIFPGDGDTDYNGKFKG (SEQ ID NO: 15), and a CDR-H3 comprising a sequence of NVFDGYWLVY (SEQ ID NO: 16), and the light chain variable region comprises CDR-L1 comprising a sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 17), a CDR-L2 comprising a sequence of QMSNLVS (SEQ ID NO: 18) and a CDR-L3 comprising a sequence of AQNLELPYT (SEQ ID NO: 19). Table 1. Complementary Determining Region (CDR) Sequences

[0077] The skilled artisan will appreciate that in Table 1, each row represents CDRs from each of the antibodies named in the left column.

[0078] The disclosure provides AAV vectors comprising sequences encoding CD20 antigen binding domains. In some embodiments, the CD20 antigen binding domain comprises a heavy chain variable region and a light chain variable region. For example, CD20 antigen binding domains that are full length antibodies or scFv comprise heavy chain variable regionsand light chain variable regions. Exemplary heavy chain variable region and a light chain variable region sequences are provided in Table 2, below.

[0079] In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, 22 or 24, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, 22 or 24. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21, 23 or 25, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21, 23 or 25.

[0080] In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, and the light chain variable region comprises a sequence of SEQ ID NO: 21. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, and is encoded by a sequence of SEQ ID NO: 26, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 20, and is encoded by a sequence of SEQ ID NO: 26. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21, and is encoded by a sequence of SEQ ID NO: 27, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 21, and is encoded by a sequence of SEQ ID NO: 27.

[0081] In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 22, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 22. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 23, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99%identity thereto. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 23. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 22, and the light chain variable region comprises a sequence of SEQ ID NO: 23.

[0082] In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 24, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 24. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 25, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain variable region comprises a sequence of SEQ ID NO: 25. In some embodiments, the heavy chain variable region comprises a sequence of SEQ ID NO: 24, and the light chain variable region comprises a sequence of SEQ ID NO: 25. Table 2. Variable Heavy and Variable Light Region sequences

[0083] The disclosure provides AAV vectors comprising sequences encoding CD20 antibodies. Sequences of antibody heavy and light chains encoded by the AAV vectors of the disclosure are provided in Table 3, below.

[0084] In some embodiments, the CD20 antibody comprises a heavy chain comprising a sequence of SEQ ID NO: 28, 30, 32, 34 or 36 or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the antibody comprises a heavy chain comprising a sequence of SEQ ID NO: 28, 30, 32, 34 or 36. In some embodiments, the antibody comprises a light chain comprising a sequence of SEQ ID NO: 29, 31, 33, 35 or 37, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the antibody comprises a light chain comprising a sequence of SEQ ID NO: 29, 31, 33, 35 or 37.

[0085] In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 29, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 29. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28, and the light chain comprises a sequence of SEQ ID NO: 29. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28, and is encoded by a sequence of SEQ ID NO: 38, or a sequence having at least 80%, at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28, and is encoded by a sequence of SEQ ID NO: 38. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 29, and isencoded by a sequence of SEQ ID NO: 39, or a sequence having at least 80%, at least 90%, at least 95% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 29, and is encoded by a sequence of SEQ ID NO: 39.

[0086] In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 30, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 30. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 31, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 31. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 30, and the light chain comprises a sequence of SEQ ID NO: 31.

[0087] In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 32, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 32. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 33, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 33. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 32, and the light chain comprises a sequence of SEQ ID NO: 33.

[0088] In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 34, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 34. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 35, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 35. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 34, and the light chain comprises a sequence of SEQ ID NO: 35.

[0089] In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 36, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 36. In some embodiments, the light chain comprises a sequence ofSEQ ID NO: 37, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 37. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 36, and the light chain comprises a sequence of SEQ ID NO: 37. Table 3. CD20 Antibody Sequences

[0090] AAV vectors of the disclosure are capable of expressing the CD20 antigen binding domain in a target cell or tissue of a subject following administration. Methods of assaying the expression of the CD20 antigen binding domain will be apparent to persons of ordinary skill in the art, and include, inter alia, ELISA assays, such as bridging ELISAs, conducted on blood, serum, tissue or CSF using the methods described in Example 1 below. In those cases where the CD20 antigen binding domain comprises the CD20 antibody rituximab, the anti- rituximab antibodies MB2A4 and AbD18423 can be used to determine the presence of rituximab encoded by the AAV vectors provided herein. Further, when the CD20 antigen binding domain comprises distinct heavy and light chains, e.g. as is the case with a full-length antibody, Western blotting as described in Example 2 can be used to assay processing of the antibody. Signal Peptides

[0091] Signal peptides, such as secretion signal peptides, can be used to enhance expression of the CD20 antigen binding domains encoded by the AAV vectors described herein. Signal peptides can be located at, or near the N terminus of the antigen binding domain. For example, in the AAV vector the sequence encoding the signal peptide can be located between a sequence encoding a promoter, and the sequence encoding the antigen binding domain.Alternatively, or in addition, a signal peptide may be located at or near the C terminus of the antigen binding domain.

[0092] In embodiments where the CD20 antigen binding domain is composed of two or more polypeptides encoded by separate sequences on the AAV vector – for example, the heavy and light chains of an antibody – each polypeptide can be operably linked to a signal peptide to enhance expression of the CD20 antigen binding domain. In some embodiments, each polypeptide is operably linked to a signal peptide at its N terminus. An example of this arrangement is shown in FIG.1.

[0093] In some embodiments of the AAV vectors described herein, the vector comprises a sequence encoding at least one signal peptide operably linked to the sequence encoding the CD20 antigen binding domain.

[0094] In some embodiments, the CD20 antigen binding domain comprises a heavy chain and chain and a light chain, and the sequence encoding the heavy chain and light chain are both operably linked to sequences encoding signal peptides. In some embodiments, the sequences encoding the signal peptides are located 5’ of the sequences encoding the heavy and light chains. In some embodiments, the signal peptides are fused to the N termini of the heavy and light chains in the resulting polypeptides.

[0095] In exemplary embodiments, the AAV vector comprises, from 5’ to 3’, a sequence encoding a promoter, a sequence encoding a first signal peptide fused to a sequence encoding a heavy chain, wherein the C terminus of first signal peptide is fused to the N terminus of the heavy chain, a linker as described herein, and a sequence encoding a second a second signal peptide fused a sequence encoding a light chain, wherein the C terminus of second signal peptide is fused to the N terminus of the light chain. In alternative embodiments, the AAV vector comprises, from 5’ to 3’, a sequence encoding a promoter, a sequence encoding a first signal peptide fused to a sequence encoding a light chain, wherein the C terminus of first signal peptide is fused to the N terminus of the light chain, a linker as described herein, and a sequence encoding a second a second signal peptide fused a sequence encoding a heavy chain, wherein the C terminus of second signal peptide is fused to the N terminus of the heavy chain. In some embodiments, the first and second signal peptides comprise the same amino acid sequence, and optionally, are encoded by different nucleotide sequences. In some embodiments, the first and second signal peptides comprise different amino acid sequences.

[0096] In some embodiments, the at least one signal peptide is isolated or derived from HGH, IgKVIII, H7, GRN, IL2 or L2. In some embodiments, e.g. those embodiments where both the heavy and light chain are fused to signal peptides, the signal peptides are independentlyselected from the group of signal peptides isolated or derived from HGH, IgKVIII, H7, GRN, IL2 and L2.

[0097] Sequences of exemplary signal peptides are provided in Table 4 below. In some embodiments the signal peptide comprises a sequence of SEQ ID NO: 44-49, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments the signal peptide comprises a sequence of SEQ ID NO: 44-49.

[0098] In some embodiments, the at least one the signal peptide is isolated or derived from HGH, and comprises a sequence of MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 44), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 44. In some embodiments, the at least one signal peptide is encoded by a sequence of SEQ ID NO: 51 or 52, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

[0099] In some embodiments, the at least one signal peptide is isolated or derived from IgKVIII comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 48), or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 48. In some embodiments, the at least one signal peptide is encoded by a sequence of SEQ ID NO: 56, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

[0100] In some embodiments, the at least one the signal peptide is isolated or derived from H7 and comprises a sequence of MEFGLSWVFLVALFRGVQC (SEQ ID NO: 45), or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 45. In some embodiments, the at least one signal peptide is encoded by a sequence of SEQ ID NO: 53, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

[0101] In some embodiments, the at least one the signal peptide is isolated or derived from GRN and comprises a sequence of MWTLVSWVALTAGLVAG (SEQ ID NO: 49), or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 49. In some embodiments, the at least one signal peptide is encodedby a sequence of SEQ ID NO: 57 or 58, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

[0102] In some embodiments, the at least one the signal peptide is isolated or derived from IL2 and comprises a sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 47), or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 47. In some embodiments, the at least one signal peptide is encoded by a sequence of SEQ ID NO: 55, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

[0103] In some embodiments, the at least one the signal peptide is isolated or derived from L2 and comprises a sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 46), or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the at least one the signal peptide comprises a sequence of SEQ ID NO: 46. In some embodiments, the at least one signal peptide is encoded by a sequence of SEQ ID NO: 54, or a sequence having at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. Table 4. Exemplary Signal Peptide SequencesLinkers

[0104] In some embodiments of the AAV vectors described herein, the CD20 antigen binding domain comprises two or more polypeptides separated by linkers. In some embodiments, the CD20 antigen binding domain comprises two polypeptides separated by a linker. The linkers can include features used to express two distinct polypeptides from a single promoter, such as, for example, protease cleave sites, self-cleaving polypeptides, and internal ribosome entry sites (IRES).

[0105] In some embodiments, the CD20 antigen binding domain comprises a heavy chain and a light chain separated by a linker. In some embodiments, the linker comprises a protease cleavage site, such as a furin or matrix metalloprotease cleavage site. In some embodiments, the linker comprises a self-cleaving peptide, such as an E2A, P2A, F2A or a T2A peptide. In some embodiments, the linker comprises a protease cleavage site and a self-cleaving peptide. In some embodiments, the linker comprises a furin cleavage site and an F2A peptide, for example a linker comprising a sequence of SEQ ID NO: 64.

[0106] In some embodiments, the linker comprises a sequence of SEQ ID NO: 64. In some embodiments, the linker is encoded by a sequence of SEQ ID NO: 65, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. Table 5. Exemplary Linker SequencesPromoters

[0107] The disclosure provides AAV vectors comprising a sequence encoding a CD20 antigen binding domain operably linked to one or more regulatory sequences, such as a promoter and / or an enhancer. The CD20 antigen binding domain can be expressed in all cells of a subject which are transduced by the AAV by using a promoter that is not tissue specific, i.e., an ubiquitous promoter. Alternatively, CD20 antigen binding domain expression can be targeted to particular cell types, such as neurons, astrocytes, oligodendrocytes or immune cells, by using cell type or tissue specific promoters such as the CAG, hSyn, or GFAP. Sequences of exemplary promoters are provided in Table 6. The promoter is located 5’ of the sequence encoding the CD20 antigen binding domain. The promoter can be locatedimmediately 5’, or separated by an intervening sequence, such as a 5’ untranslated region (5’ UTR). Additional promoters suitable for inclusion in the AAV vectors described herein will be known to persons of ordinary skill in the art.

[0108] In some embodiments, the promoter is capable of expressing the CD20 antigen binding domain in a central nervous system cell, an immune cell, or a cell derived from a central nervous system cell or immune cell. In some embodiments, the central nervous system cell comprises a brain cell and / or spinal cord cell. In some embodiments, the central nervous system cell comprises an astrocyte, an oligodendrocyte or a neuron. In some embodiments, the immune cell is a B-lymphocyte, or a precursor or derivative thereof. In some embodiments, the cell derived from the immune cell is a primary central nervous system lymphoma (PCNSL) cell or a secondary central nervous system lymphoma cell.

[0109] In some embodiments, the promoter drives expression in the CNS. In some embodiments, the promoter drives expression in neuronal cells or glial cells. In some embodiments, the promoter that drives expression in glial cells is a GFAP promoter, gfaABC1D promoter, gfa2 promoter, ALDH1L1 promoter, SLC1A3 promoter, Gjb6 promoter, Mbp promoter, MAG promoter, CBh promoter, F4 / 80 promoter, CD68 promoter, or CD11B promoter. In some embodiments, the promoter that drives expression in neuronal cells is a neuronal-specific enolase (NSE) promoter, Synapsin 1 promoter, calcium / calmodulin- dependent protein kinase II promoter, tubulin alpha 1 promoter, platelet- derived growth factor beta chain promoter, parvalbumin promoter, GAD67 promoter or CCK promoter. In some embodiments, the promoter is a ubiquitous promoter. Exemplary ubiquitous promoters include the major immediate early human cytomegalovirus promoter (MIEhCMV), Chicken ȕ-Actin Promoter (CBA), Human Cytomegalovirus Immediate / Early Gene Promoter and Enhancer (CMV), Chicken ȕ-Actin / Cytomegalovirus Hybrid Promoter (CAG), Rous Sarcoma Virus Long Terminal Repeat Promoter (RSV), SV40 promoter or EF1alpha promoter.

[0110] The AAV vector can also include an enhancer, such as a CMV Enhancer, mDlx enhancer, or an AQP4 enhancer. Suitable enhancers will be known to persons of ordinary skill in the art.

[0111] In some embodiments, the promoter is selected from the group consisting of a CAG promoter, an hSyn promoter, a GFAP promoter, and a CBA promoter.

[0112] In some embodiments, the CAG promoter comprises a sequence of SEQ ID NO: 40, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% orat least 99% identity thereto. In some embodiments, the CAG promoter comprises a sequence of SEQ ID NO: 40.

[0113] In some embodiments, the hSyn promoter comprises a sequence of SEQ ID NO: 41, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the hSyn promoter comprises a sequence of SEQ ID NO: 41.

[0114] In some embodiments, the GFAP promoter comprises a sequence of SEQ ID NO: 42, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the GFAP promoter comprises a sequence of SEQ ID NO: 42.

[0115] In some embodiments, the CBA promoter comprises a sequence of SEQ ID NO: 43, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the CBA promoter comprises a sequence of SEQ ID NO: 43. Table 6. Exemplary Promoter SequencesNon-coding Sequences

[0116] AAV vectors of the disclosure can include additional non-coding sequences to either enhance expression of the CD20 antigen binding domain in target cells, such as central nervous system cells, or reduce expression of the CD20 antigen binding domain in non-target cells (e.g., liver, and / or antigen presenting cells). Such sequences can be located 5’ of thesequence encoding the CD20 antigen binding domain in the vector (e.g., in a 5’ untranslated region, or 5’ UTR), or 3’ of the sequence encoding the CD20 antigen binding domain (e.g., in a 3’ UTR), or a combination thereof.

[0117] Exemplary non-coding sequences include microRNA target sites that can be incorporated into the expression vector to reduce expression in the liver, and increase the brain to periphery ratio of expression of the encoded CD20 antigen binding domain. Alternatively, or in addition, microRNA target sites can be incorporated into the expression vector that reduce expression by antigen-presenting cells (APCs). Without wishing to be bound by theory, it is thought that this can reduce immunogenicity of the CD20 antigen binding domain expressed from the AAV vector.

[0118] Exemplary sequences encoding microRNA targeting sequences are provided in Table 7 below. The microRNA sequences of the disclosure are preferably included in a 3’ untranslated region, i.e., a sequence 3’ of the sequence encoding the CD20 antigen binding domain.

[0119] In some embodiments, the polynucleotide encoding the CD20 antigen binding domain comprises one or more microRNA (miRNA) targeting sequences that reduce expression of the CD20 antigen binding domain in the liver and / or antigen present cells (APC) of the subject when compared to an AAV vector that does not include the one or more miRNA targeting sequences. In some embodiments, the one or more microRNA targeting sequences comprise a miR122 sequence, a miR-142-3p sequence, or a combination thereof. In some embodiments, the miRNA targeting site comprises a sequence selected from the group consisting of SEQ ID NOS: 59-61. In some embodiments, the one or more miRNA targeting sequences are 3’ of the sequence encoding the CD20 antigen binding domain, for example in a 3’ UTR. Table 7. microRNA Targeting Sequences

[0120] The AAV vectors of the disclosure may optionally include other regulatory elements that may be used to enhance expression of the CD20 antigen binding domain encoded by the vector. For example, AAV vectors as described herein can include a sequence of a Woodchuck Hepatitis Posttranscriptional Regulatory Element (WPRE), as described in Table 8 below. In some embodiments, the WPRE comprises a sequence of SEQ ID NO: 62, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the WPRE comprises a sequence of SEQ ID NO: 62. In some embodiments, the sequence of the WPRE is 3’ of the sequence encoding the CD20 antigen binding domain. In some embodiments, for example those embodiments where the vector comprises one or more microRNA targeting sequences, the one or more microRNA targeting sequences are located 3’ of the sequence encoding the CD20 antigen binding domain, followed by the WPRE.

[0121] In some embodiments, the AAV vector comprises a polyadenylation (polyA) signal 3’ of the sequence encoding the CD20 antigen binding domain. Exemplary polyA signals are provide in table 8 below and include the SV40 polyA, hGH, bGH and rbGlob polyA signals.

[0122] In some embodiments, the polyadenylation signal comprises a sequence of SEQ ID NO: 63, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto. In some embodiments, the polyadenylation signal comprises a sequence of SEQ ID NO: 63.

[0123] In some embodiments, the AAV vector comprises a 3’ UTR comprising, from 5’ to 3’, one or more microRNA targeting sites comprising a sequence set forth in Table 7, a WPRE, and a polyA signal comprising a sequence set forth in Table 8. Alternatively, the AAV vector may comprise, 3’of the sequence encoding the CD20 antigen binding domain, the WPRE followed by the polyA signal. As a still further alternative, the AAV vector may comprise, 3’of the sequence encoding the CD20 antigen binding domain, the polyA signal, omitting either one or more microRNA targeting sites, the WPRE, or both.Table 8. Expression Elements

[0124] AAV vectors of the disclosure generally comprise inverted terminal repeats (ITRs) at either of end of the polynucleotide (the viral genome) that is packaged into the AAV vector.

[0125] In some embodiments, the polynucleotide comprises, from 5’ to 3’: an 5’ AAV inverted terminal repeat (ITR), optionally an enhancer, a promoter, a sequence encoding a signal peptide, a sequence encoding a CD20 antigen binding domain, a sequence encoding a 3’ UTR, and a 3’ AAV ITR. In some embodiments, the 3’ UTR comprises, from 5’ to 3’, one or more microRNA targeting sites, a WPRE, and a polyA signal. In alternative embodiments, the 3’ UTR comprises a WPRE and a polyA signal. In further alternative embodiments, the 3’ UTR comprises a polyA signal.

[0126] In some embodiments, the polynucleotide comprises, from 5’ to 3’: an 5’ AAV ITR, optionally an enhancer, a promoter, a sequence encoding a first signal peptide, a sequence encoding a CD20 antigen binding domain heavy chain, a sequence encoding a linker comprising one or more protease cleavage sites and / or self-cleaving peptides, or an internal ribosome entry site (IRES), a sequence encoding a second signal peptide, a sequence encoding a CD20 antigen binding domain light chain, and a 3’ AAV2 ITR. In some embodiments, the polynucleotide comprises, from 5 to 3’: an 5’ AAV ITR, optionally an enhancer, a promoter, a sequence encoding a first signal peptide, a sequence encoding a CD20 antigen binding domain light chain, a sequence encoding a linker comprising one or more protease cleavage sites and / or self-cleaving peptides, or an internal ribosome entry site (IRES), a sequence encoding a second signal peptide, a sequence encoding a CD20 antigen binding domain heavy chain and a 3’ AAV2 ITR. In some embodiments, the polynucleotide comprises, between the sequence encoding the CD20 antigen binding domain light chain or heavy chain (depending on configuration) and the 3’ AAV2 ITR, one or more microRNA targeting sequences, a WPRE and a polyadenylation signal. In some embodiments, the polynucleotide comprises, between the sequence encoding the CD20 antigen binding domain light chain or heavy chain (depending on configuration) and the 3’ AAV2 ITR, a WPRE and a polyadenylation signal. In some embodiments, the polynucleotide comprises, between thesequence encoding the CD20 antigen binding domain light chain or heavy chain (depending on configuration) and the 3’ AAV2 ITR, a polyadenylation signal. AAV Vectors

[0127] The disclosure provides AAV vectors comprising a capsid, and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain. The AAV capsid may be a commonly used AAV capsid (e.g., AAV9 or rh10) or an engineered CNS-tropic AAV capsid, e.g. as described in WO 2022 / 272240, the contents of which are incorporated herein by reference.

[0128] AAV is a tiny non-enveloped virus having a 25 nm capsid. No disease is known or has been shown to be associated with the wild-type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been shown to exhibit long-term episomal transgene expression, and AAV has demonstrated excellent transgene expression in the brain, particularly in neurons. Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.7 kb. An AAV vector such as that described in Tratschin et al., Mol. Cell. Biol.5:3251-3260 (1985) can be used to introduce DNA into cells. There are numerous alternative AAV variants (over 100 have been cloned), and AAV variants have been identified based on desirable characteristics.

[0129] In some embodiments, the AAV include targeting peptides that enhance permeation through the blood brain barrier (BBB), e.g., when inserted into the capsid of an AAV, e.g., AAV1, AAV2, AAV8, or AAV9, or when conjugated to a biological agent, e.g., an antibody or other large biomolecule, either chemically or via expression as a fusion protein. In some embodiments, the targeting peptides comprise sequences of at least 5 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, contiguous amino acids of the sequences VPALR or VSALK. In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5, wherein: (i) X1, X2, X3, X4 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and (ii) X5 is K, R, H, D, or E. In some embodiments, the targeting peptides comprise sequences of at least 6 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5 or 6 contiguous amino acids of the sequences TVPALR (SEQ ID NO: 91), TVSALK (SEQ ID NO: 92), TVPMLK (SEQ ID NO: 100) and TVPTLK (SEQ ID NO: 101). In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6, wherein: (i) X1 is T; (ii) X2, X3, X4, X5 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and (iii) X6 is K, R, H, D, or E. In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6,wherein: (i) X1, X2, X3, X4 are any four non-identical amino acids from V, A, L, I, G, P, S, T, or M; (ii) X5 is K, R, H, D, or E; and (iii) X6 is E or D. In some embodiments, the targeting peptides comprise sequences of at least 7 amino acids. In some embodiments, the amino acid sequence comprises at least 4, e.g., 5, 6, or 7 contiguous amino acids of the sequences FTVSALK (SEQ ID NO: 93), LTVSALK (SEQ ID NO: 94), TVSALFK SEQ ID NO: 96), TVPALFR (SEQ ID NO: 97), TVPMLFK (SEQ ID NO: 98) and TVPTLFK (SEQ ID NO: 99). In some other embodiments, the targeting peptides comprise a sequence of X1X2X3X4X5X6X7, wherein: (i) X1 is F, L, W, or Y; (ii) X2 is T; (iii) X3, X4, X5, X6 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; and (iv) X7is K, R, H, D, or E. In some embodiments, the targeting peptides comprise a sequence of X1X2X3X4X5 X6 X7, wherein: (i) X1 is T; (ii) X2, X3, X4, X5 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; (iii) X6 is K, R, H, D, or E; and (iv) X7 is E or D. In some embodiments, the targeting peptides comprise a sequence of X1 X2 X3 X4 X5 X6 X7, wherein: (i) X1, X2, X3, X4 are any four non-identical amino acids of V, A, L, I, G, P, S, T, or M; (ii) X5 is K, R, H, D, or E; (iii) X6 is E or D; and (iv) X7 is A or I. In some embodiments, the targeting peptides comprise a sequence of V[S / p][A / m / t / ]L, wherein the upper-case letters are preferred at that position. In some embodiments, the targeting peptides comprise a sequence of TV[S / p][A / m / t / ]L. In some embodiments, the targeting peptides comprise a sequence of TV[S / p][A / m / t / ]LK (SEQ ID NO: 173). In some embodiments, the targeting peptides comprise a sequence of TV[S / p][A / m / t / ]LFK (SEQ ID NO: 174). In some embodiments, the targeting peptide does not consist of VPALR (SEQ ID NO: 89) or VSALK (SEQ ID NO: 90). In some embodiments, the peptide insert comprises TVSALFK (SEQ ID NO: 96). In some embodiments, the peptide insert comprises TVSALK (SEQ ID NO: 92). In some embodiments, the peptide insert consists of TVSALFK (SEQ ID NO: 96). In some embodiments, the peptide insert consists of TVSALK (SEQ ID NO: 92). Exemplary targeting peptides are provided in Table 9, below. Table 9. Targeting peptides

[0130] Targeting peptides including reversed sequences can also be used, e.g., KLASVT (SEQ ID NO: 159) and KFLASVT (SEQ ID NO: 160). Preferred AAV for use in the present methods and compositions include AAV.CPP.16 and AAV.CPP.21 (described in WO 2020 / 014471). Other AAV as known in the art (e.g., AAV1, 2, 3, 4, 5, 6, 7, 8 and variants thereof and others as known in the art or described herein) can also be used. AAV.CPP.16 can be used for targetingCNS, liver, heart / muscle, and / or lung. In some embodiments, the AAV vector comprises an AAV9 or rh10 capsid. In some embodiments, the capsid comprises a targeting peptide, i.e., the capsid is a recombinant capsid. In some embodiments, the capsid comprises an AAV9 capsid with a targeting peptide inserted between amino acids 588 and 589 of SEQ ID NO: 1. In some embodiments, the targeting peptide comprises TVSALFK (SEQ ID NO: 96) or TVSALK (SEQ ID NO: 92).

[0131] Exemplary VP1 capsid protein sequences for AAV9, AAV.CPP.16 and AAV.CPP.21 are provided in Table 10 below: Table 10. Exemplary Capsid VP1 sequences

[0132] In some embodiments, the AAV vectors of the disclosure comprise a polynucleotide comprising a sequence encoding a CD20 antigen binding domain, and an capsid comprising a sequence of SEQ ID NO: 1, 66 or 67, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto, wherein an AAV comprising the capsid is capable of crossing the blood brain barrier to a degree that is equivalent to an AAV comprising a capsid comprising a sequence of SEQ ID NO: 1, 66 or 67. In some embodiments, the AAV vectors of the disclosure comprise a polynucleotide comprising a sequence encoding a CD20 antigen binding domain, and an capsid comprising a sequence of SEQ ID NO: 1, 66 or 67.

[0133] Any suitable ITRs may be used with the AAV vectors of the instant disclosure. In some embodiments, the AAV vector comprises an AAV9 capsid (or a derivative thereof in which the capsid comprises a targeting peptide) and AAV2 ITRs. An exemplary AAV25’ ITR for use with the AAV vectors of the disclosure comprises a sequence of CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGG CGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGG AGTGGCCAACTCCATCACTAGGGGTTCCT (SEQ ID NO: 157). An exemplary AAV23’ ITR sequence comprises AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTG AGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTG AGCGAGCGAGCGCGCAGCTGCCTGCAGG (SEQ ID NO: 158). However, the skilled artisan will understand that suitable ITR sequences are available from a variety of sources. Pharmaceutical Compositions

[0134] The disclosure provides pharmaceutical compositions comprising the AAV vectors described herein and a pharmaceutically acceptable carrier, diluent or excipient.

[0135] As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration.Examples of routes of administration include parenteral, e.g., intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion administration. Further exemplary routes of administration include intrathecal administration (e.g., intracerebroventricular injection, intracisterna magna injection or injection into the subarachnoid space of the spinal cord). Delivery can thus be systemic or localized.

[0136] Methods of formulating suitable pharmaceutical compositions are known in the art, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and the books in the series Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, solutions or suspensions used for parenteral administration can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. As a further example, solutions or suspensions used for intrathecal administration can include a suspension of AAV vectors in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant and optional excipients. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral or intrathecal preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous or intrathecal administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).

[0137] In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and bythe use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0138] Sterile injectable solutions can be prepared by incorporating the AAV vectors in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the AAV vector into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Polynucleotides

[0139] The disclosure provides polynucleotides comprising sequences encoding an AAV viral genome comprising a sequence encoding CD20 antigen binding domain as described herein.

[0140] In some embodiments, the polynucleotide comprises a promoter operably linked, i.e., 5’ and proximal to, the sequence encoding the CD20 antigen binding domain. In some embodiments, the promoter comprises a sequence of SEQ ID NO: 40-43, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the promoter comprises a sequence of SEQ ID NO: 40-43.

[0141] In some embodiments, the CD20 antigen binding domain comprises a heavy chain variable region and a light chain variable region. As a non-limiting example, CD20 antigen binding domains that are scFv contain a heavy chain variable region and a light chain variable region fused by a linker. In such embodiments, the polynucleotide comprises sequences encoding the heavy chain variable region and the light chain variable region, for example sequences of SEQ ID NO: 26 and SEQ ID NO: 27, respectively, or sequences having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the CD20 antigen binding domain is fused to a signal peptide, optionally the N terminus of the CD20antigen binding domain is fused to the signal peptide. In some embodiments, the sequence encoding the signal peptide is selected from the group consisting of SEQ ID NOS: 51-58.

[0142] In some embodiments, the CD20 antigen binding domain comprises an antibody, and the antibody comprises a heavy chain and a light chain. In such embodiments, the polynucleotide comprises sequences encoding the heavy chain and the light chain. In some embodiments, the heavy chain comprises a sequence of SEQ ID NO: 28, and is encoded by a polynucleotide comprising a sequence of SEQ ID NO: 38, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the light chain comprises a sequence of SEQ ID NO: 29, is encoded by a polynucleotide comprising a sequence of SEQ ID NO: 39, or a sequence having at least 80%, at least 90%, at least 95%, or at least 99% identity thereto. In some embodiments, the heavy chain and the light chain are both fused to a signal peptide, optionally at the N terminus. In some embodiments, the sequences encoding the signal peptide fused to the heavy chain and the signal peptide fused to the light chain are independently selected from the group consisting of SEQ ID NOS: 51-58. In some embodiments, the heavy chain and the light chain are separated by a linker as described herein, for example a linker encoded by the sequence of SEQ ID NO: 65.

[0143] In some embodiments, the polynucleotide encoding the AAV viral genome comprises one or more additional sequence elements located 3’ to (or after the C-terminus of) the CD20 antigen binding domain. In some embodiments, the additional sequence element comprises one or more microRNA targeting sequences, for example as set forth in Table 7. In some embodiments, the more microRNA targeting sequences comprise a sequence of SEQ ID NO: 59- 61. In some embodiments, the additional sequence element comprises a WPRE, for example a WPRE comprising a sequence of SEQ ID NO: 62. In some embodiments, the additional sequence element comprises a polyA signal, for example a polyA comprising a sequence as set forth in Table 8. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, a sequence of a polyA. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, sequences of a WPRE and a polyA. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, sequences of one or more microRNA binding sites, a WPRE and a polyA. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, a polyA comprising a sequence ofSEQ ID NO: 63. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, a WPRE comprising a sequence of SEQ ID NO: 62, and a polyA comprising a sequence of SEQ ID NO: 63. In some embodiments, the polynucleotide comprises, 3’ to the sequence encoding the CD20 antigen binding domain, one or more microRNA binding sites selected from the group consisting of SEQ ID NO: 59-61, a WPRE comprising a sequence of SEQ ID NO: 62, and a polyA comprising a sequence of SEQ ID NO: 63.

[0144] In some embodiments, the polynucleotide encoding the AAV viral genome comprises, from 5’ to 3’, a 5’ inverted terminal repeat (ITR), for example an AAV25' ITR as described herein, a sequence encoding a promoter, a sequence encoding a CD20 antigen binding domain, one or more microRNA targeting sequences, and a 3’ ITR, for example an AAV23' ITR as described herein. In some embodiments, the CD20 antigen binding domain is encoded by a sequence comprising, from 5’ to 3’: a sequence encoding a first signal peptide selected from the group consisting of SEQ ID NOS: 51-58; a sequence encoding a heavy chain, for example SEQ ID NO: 38; a sequence encoding the linker, for example SEQ ID NO: 65; a sequence encoding a second signal peptide selected from the group consisting of SEQ ID NOS: 51-58; and a sequence encoding the light chain, for example SEQ ID NO: 39.

[0145] The disclosure provides polynucleotides encoding the Rep and Cap proteins described herein, for example the capsid proteins comprising targeting sequences described supra. In some embodiments, the polynucleotide comprises a sequence encoding a capsid comprising a VP1 comprising an amino acid sequence of SEQ ID NO: 1, 66 or 67.

[0146] In some embodiments, the polynucleotides provided herein are included in vectors, example for transforming cells, or for recombinant DNA manipulation. Suitable vectors include plasmids, viral vectors, cosmids and the like, the selection of which is within the skill of the person of ordinary skill in the art. Methods of Treatment

[0147] The disclosure provides methods of treating a cancer in a subject, comprising administering an effective amount of the AAV vectors or the pharmaceutical compositions described herein to the subject.

[0148] In some embodiments, the methods comprise administering a therapeutically effective amount of a composition comprising an adeno-associated viral (AAV) vector comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain. In some embodiments, the AAV vector comprises one or more modifications to the capsid which increase the efficiency with which the AAV vector crosses the blood brain barrier relative to an AAV vector comprising an unmodified capsid. In some embodiments, the one or more modifications comprises a targeting peptide in the capsid, for example one of the targeting peptides described supra. The CD20 antigen binding domain can be any of the CD20 antigen binding domains described supra. For example, the CD20 antigen binding domain comprises an antibody comprising a heavy chain comprising a sequence of SEQ ID NO: 28, and a light chain comprising a sequence of SEQ ID NO: 29.

[0149] In some embodiments, the cancer is a cancer of the central nervous system. In some embodiments, the cancer comprises primary central nervous system lymphoma (PCNSL) or secondary central nervous system lymphoma. In some embodiments, the cancer comprises cells that express CD20, such that the CD20 antigen binding domain encoded by the AAV vectors described herein binds to the cells of the cancer.

[0150] The AAV vectors or pharmaceutical compositions may be administered by any suitable route of administration. Administration may be systemic, for example via intravenous injection or infusion. Administration may be parental. Administration may be intrathecal, i.e., into the cerebrospinal fluid (CSF).

[0151] Exemplary routes of intrathecal administration include intracerebroventricular (ICV) injection, intracisterna magna (ICM) injection or injection into the subarachnoid space of the spinal cord (IT). Intrathecal administration may comprise introducing the AAV vector or pharmaceutical composition into a cerebral ventricle. Alternatively, intrathecal administration may comprise introducing the pharmaceutical composition into the lower back. Intrathecal delivery may include lumbar puncture, intraventricular, suboccipital / intracisternal, and / or C l-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 a sterna magna. Such administration can be by means of a bolus injection. In other embodiments, intrathecal administration is achieved through the use of an infusion pump.

[0152] In some embodiments, the AAV vectors or pharmaceutical compositions described herein are administered once. In some embodiments, the AAV vectors or pharmaceutical compositions are administered more than once, for example 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times. Suitable volumes for administration can be determined based on concentration of the AAV vector, route of administration, and target dose.

[0153] In some embodiments, the AAV vectors or pharmaceutical compositions described herein are administered as a monotherapy.

[0154] In some embodiments, the AAV vectors or pharmaceutical compositions described herein are administered as part of a combination therapy. For example, the AAV vectors or pharmaceutical compositions may be administered in combination with a chemotherapy such as CHOP (a chemotherapy combination including cyclophosphamide, doxorubicin (Adriamycin) vincristine (Oncovin)) or HD-MTX (high dose methotrexate). Alternatively, or in addition, the AAV vectors or pharmaceutical compositions can be administered in combination with therapies such as whole brain radiotherapy or autologous stem cell-based therapies such as chimeric antigen receptor T cell (CAR-T) therapies.

[0155] In some embodiments, administration of the AAV vectors or pharmaceutical compositions can reduce a sign or a symptom of the cancer, slow or halt the progression of the cancer; reduces size and / or number of tumors of the cancer, and / or increases survival of the subject.

[0156] For example, administration of the AAV vectors or pharmaceutical compositions may result in a reduction in size of a tumor. A reduction in size of a tumor may also be referred to as “tumor regression.” Preferably, after administration, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0157] Administration of the AAV vectors or pharmaceutical compositions may result in a reduction in tumor volume. Preferably, after administration, tumor volume is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor volume is reduced by 10%or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75% or greater. Tumor volume may be measured by any reproducible means of measurement.

[0158] Administration of the AAV vectors or pharmaceutical compositions may result in a decrease in number of tumors. Preferably, after administration, tumor number is reduced by 5% or greater relative to number prior to treatment; more preferably, tumor number is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0159] Administration of the AAV vectors or pharmaceutical compositions may result in a decrease in number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after administration, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, 10x, or 50x.

[0160] Administration of the AAV vectors or pharmaceutical compositions can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone, or a population receiving an alternative therapy. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment withthe AAV vector or pharmaceutical composition. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with the AAV vector or pharmaceutical composition.

[0161] Administration of the AAV vectors or pharmaceutical compositions can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone, or receiving an alternative therapy. Administration of the AAV vector or pharmaceutical composition can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Administration of the AAV vector or pharmaceutical composition can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not an AAV vector or pharmaceutical composition of the disclosure. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with the AAV vector or pharmaceutical composition.

[0162] Administration of the AAV vectors or pharmaceutical compositions can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number prior to treatment; more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 50%; and most preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0163] Administration of the AAV vectors or pharmaceutical compositions can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 50%; and most preferably, less than 75%. Tumor regrowth may bemeasured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by failure of tumors to reoccur after treatment has stopped. Methods of Making AAV Vectors

[0164] Methods of making the AAV vectors of the disclosure will be apparent to persons of ordinary skill in the art. In general, the methods include producing a polynucleotide sequence encoding the CD20 antigen binding domain and the flanking ITRs, and incorporating this polynucleotide into an expression vector appropriate for expression by a packaging cell line. This expression vector is then used to transform an appropriate packaging cell line, which expresses the AAV Rep and Cap genes, as well as appropriate helper genes from other adeno or herpes viruses. The packaging cell is then cultured under conditions sufficient to produce the AAV, which are recovered by methods known in the art. Rep and Cap can be provided to the packaging cells as plasmids, or, as an alternative, be stably integrated into the packaging cell genome. Suitable packaging cell lines are known to one of ordinary skill in the art and include, but are not limited to HEK293, Huh-7, HeLa, HepG2, Hep1A, CHO, COS, NIH3T3 and the like. In an exemplary protocol, HEK293 cells are transfected with either two or three plasmids: one encoding the CD20 antigen flanked by AAV ITRs, one carrying the AAV rep / cap genes, and another containing helper genes.

[0165] Methods of purifying AAV produced by host cell lines will be known to one of ordinary skill in the art, and include, without limitation, depth filtration, affinity chromatography, gradient centrifugation, ion exchange chromatography and combinations thereof. Standard recombinant techniques in molecular biology can be used to make the polynucleotides and AAV vectors of the present disclosure.

[0166] In some embodiments, making the AAV vectors of the disclosure comprises contacting a packaging cell with a polynucleotide comprising a sequence encoding a CD20 antigen binding domain flanked by AAV ITRs, culturing the packaging cell under conditions sufficient to produce the AAV vector, and purifying the AAV vector from the cell and / or cell culture medium. In some embodiments, the methods comprise contacting the cell with one or more polynucleotides comprising sequences encoding the Rep and Cap genes, and helper genes. Insome embodiments, the contacting comprises transfection. In alternative embodiments, the contacting comprises stable transformation, e.g., to produce a packaging cell line expressing Rep and Cap. Kits and Articles of Manufacture

[0167] The disclosure provides comprising the AAV vectors and pharmaceutical compositions described herein. In some embodiments, the kit comprises instructions for use. In some embodiments, the AAV vector and / or pharmaceutical composition is packaged in a tube, vial, syringe, ampoule or the like. EXAMPLES Example 1: In vitro Transfection of Cells with AAV Expressing CD20 Antibody

[0168] AAV vectors encoding the CD20 antibody heavy and light chains from rituximab were designed according to the diagram shown in FIG.1. The vectors included AAV2 ITR sequences, a 5’ promoter, followed by a sequence encoding the rituximab heavy chain linked to a first signal peptide at its N-terminus. The heavy chain sequence was separated from the sequence encoding the light chain, also linked to an N-terminal signal peptide, by a linker that included a furin cleavage site and an F2A self-cleaving peptide. 3’ to the cassette encoding the CD20 antibody, the vectors also optionally included microRNA targeting sites to reduce expression in the liver and / or antigen presenting cells (APCs), optionally a WPRE, and a polyadenylation signal. The choice of promoter, signal peptide, microRNA (or absence thereof) and presence or absence of WPRE in each vector construct tested are shown in FIG.2. Sequences are provided in Tables 1- 8, above. Full length sequences (from ITR to ITR, excluding cloning vector backbone) are provided at the end of the Examples.

[0169] To test expression of rituximab from the vectors, 293T cells were transiently transfected in 12-well plates with the expression vectors listed in FIG. 2 at 1 ug of DNA per well. 48 hours post-transfection, media from the cells were collected, and rituximab concentration in the media was measured using rituximab-specific ELISA assay. Reagents for the ELISA assay are provided in Table 1-1 below. In brief, coat plates were coated with rituximab antibody (AbD18423) by adding 20 μL (of a 0.5mg / mL stock) to 10 mL of coating buffer (per plate). The plates were allowed to sit for 1 hour at room temperature (approximately 22-25 ^C). After coating, plateswere washed three times in a standard ELISA wash buffer, and blocked with ELISA blocking buffer for thirty minutes.

[0170] After blocking, a CD20 antibody control and the media samples were added. As a control, serial dilutions of a CD20 antibody (10F381 - Rituximab Biosimilar) were used.10F381 at a 1 mg / mL working stock was diluted to 500 ng / mL immediately before use.20 μL of the 500 ng / mL was added to a well containing 40 μL of sample diluent and pipetted up and down to mix, 20 μL of the resulting mixture was added to a subsequent well containing 40 μL of sample diluent, and this was repeated 3X to generate the serial 10F381 dilutions. For the samples of transfected cell supernatants, a 1:100 dilution was used as starting point. 50μL of the initial sample was added to 450μL of sample diluent to make a 1:100 Sample Diluent.40 μL of the 1:100 Sample Diluent was added to an initial row of the sample columns, and diluted 1:5 in subsequent rows by taking 10 μL from the first row, adding it to 40 μL of sample diluent in the next row and repeating.

[0171] Samples were allowed to incubate for 90 minutes. Following incubation, the plates were washed 5x to remove the sample, and 100 μL / well of the detection antibody was added. For anti- CD20 ELISAs, the anti-rituximab antibody MB2A4 conjugated to HRP was used, as if samples have any kind of human IgG it could interfere with the readout (i.e., human matrices, such as human serum or CSF). This antibody was used in a 1:1,000 dilution, in sample diluent. Samples were incubated with the detection antibody for 60 minutes at room temperature, washed three times, and then incubated with 100 μL / well of 1:1 proportion of TMB Peroxidase Substrate + Peroxidase Substrate Solution B (TMB) prepared 10 minutes before use. The plates were allowed to sit until the standard showed a range of colors, usually about 20 minutes.100 μL / well of 3M H2SO4was then used to stop the reaction. Plates were read in an ELISA plate reader within 15 minutes of stopping the reaction.

[0172] Data from medium from 293T cells were transiently transfected with constructs 1-11 is shown in FIG.3. As can be seen from FIG. 3, 9 of the 11 vector constructs expressed CD20 antibody in the transient transfection assay. Table 1-1. Reagents for quantification or Rituximab by ELISAExample 2: Expression and Processing of Rituximab Heavy and Light Chains by Transiently Transfected 293T cells

[0173] Western blot analysis was used to verify correct expression and processing of rituximab heavy and light chains from the transiently transfected 293T cells described in Example 1.

[0174] Rituximab-containing media from 293T cells transfected with rituximab expression vectors were analyzed using a Western blot with anti-human IgG1 detection antibodies as described below.

[0175] Total protein in the cell culture medium, and target protein concentration, was quantified prior to analysis using the BCA protein assay for total protein and ELISA for rituximab (as described in Example 1). Reagents for Western blotting are shown in Table 2-1 below.

[0176] Samples were diluted such that 10 μL contained a similar concentration of the target protein. Generally, a concentration of 10 μg / mL of the target protein was used, and around 100 ng target protein per well was loaded on the gel.

[0177] For reducing gels, 8 μL of each sample was mixed with 1 μL of 10x Reducing agent and 4 μL LDS Sample Buffer. For non-reducing gels, 9 μL of each sample was mixed with 4 μL of LDS Sample Buffer. Samples were incubated at 70 ºC for 10 to 15 minutes.

[0178] After incubation, samples were run on a Mini Protein Gel in the Mini Gel Tank filled with 1X MOPS buffer at 200V for 32 minutes. 5μL of PageRuler prestained ladder was added to the first and last lanes, and 10 μL of heated sample was added to each ladder.10F381 (rituximab Biosimilar) was included as a positive control, as was a negative control. In general, 100 ng of positive control was used for rituximab.

[0179] After running the gel, proteins were transferred from the gel to a membrane with an iBlotTM2 gel transfer device according to standard methods. After transfer, the membrane was cut to the area where the gel was transferred, and rinsed 3X for 1 minute with dH2O.

[0180] In some cases, membranes were stained with Ponceau stain to evaluate transfer efficiency.

[0181] Blotting was performed by first incubating the membrane in 1X BSA blocking solution for 2 hours at room temperature, while shaking it on an orbital shaker at 70 rpm. After blocking, the membrane was washed 5X with Blotting Wash buffer, shaking for 5 minutes for each wash. After washing, the membrane was incubated with the primary-HRP conjugated antibody diluted at 1:1,000 in 1X BSA blocking solution. The membrane was incubated with primary antibody either 2 hours at room temperature, or overnight at 4q C. Next, the membrane was washed 5X with Blotting Wash buffer, shaking for 5 minutes for each wash, then 3X with dH20, shaking for 1 minute for each wash. Finally, the membrane was incubated with ECL Western Blotting Substrate (about 5 to 10 minutes) and then read on a Chemiluminescence reader.

[0182] The results are shown in FIG. 4. As can be seen in FIG.4, heavy and light chains are clearly visible for the majority of vectors, indicating correct processing of the IgG1 subunits. Table 2-1. Western Blotting ReagentsExample 3: In vivo Testing of Rituximab AAV Expression Vectors in Mice

[0183] AAV vectors were generated by packaging the rituximab expression vectors listed in FIG.2 into the BRV16 capsid using baculoviral Sf9 AAV packaging system according to standard methods. The BRV16 capsid system is described in US2023 / 0053817. In brief, a cell penetrating peptide TVSALK (SEQ ID NO: 92) is inserted into the AAV9 capsid VP1 protein (SEQ ID NO: 1) between amino acid residues 588 and 589, generating the VP1 capsid of SEQ ID NO: 66.

[0184] Adult female wild-type mice were treated intravenously with a single AAV vector dose of 5e13 viral genomes per kg. Tissues (brain and peripheral organs) and plasma were collected 20 days post-dosing. Rituximab expression was quantified in the tissues and plasma using the ELISA assay described above in Example 1.

[0185] Levels of rituximab in plasma, brain, muscle and liver are shown in FIGS.5A-5D. As can be seen in FIG. 5B, constructs BRV-P-001, BRV-P-002, BRV-P-003, BRV-P-004, BRV-P-005, BRV-P-006, BRV-P-007, BRV-P-009, and BRV-P-011 show expression in brain tissue, with the highest levels shown by BRV-P-001, BRV-P-006 and BRV-P-007. Example 4: Efficacy of Rituximab Expressing AAV in Mouse Models of Primary CNS Lymphoma (PCNSL)

[0186] A single dose of a rituximab AAV vector is administered via systemic (IV), intra-CSF (intra-cisterna magna, intra-cerebroventricular, or intrathecal) or intra-cerebral route at a single dose in one of the following mouse models of PCNSL, described in Table 4-1 below. Table 4-1 PCNSL mouse models with validated efficacy response to intra-cerebrally administered rituximab

[0187] Anesthetized mice from the indicated mouse strain (BALB / c (H-2d) or C3H / HeN (H-2k)) are injected into the cerebral striatum or the vitreous (e.g., through the pars plana or in the caudate nucleus) with an appropriate volume of cells to implant a tumor of the indicated cell line (e.g., 5 × 104 cells in a final volume of 2 ^L). A single dose of AAV rituxumab vector is administered, as described above, and tumor volume, body weight and metastasis in AAV rituximab injected mice versus control mice is monitored. Vector Sequences BRV-P-001 (SEQ ID NO: 162) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG CGACGGGTTC AAGAACTTCC CTACTTCTTG 1261 CATTTGGCCT GCTTTGTTTG CCGTGGTTAC AGGAGGGCTC GGCACAGGTT CAGCTGCAAC 1321 AGCCTGGCGC CGAACTTGTG AAACCTGGCG CCTCTGTGAA GATGAGCTGC AAGGCCAGCG 1381 GCTACACCTT CACCAGCTAC AACATGCACT GGGTCAAGCA GACCCCTGGC AGAGGCCTGG 1441 AATGGATCGG AGCCATCTAT CCCGGCAACG GCGACACCTC CTACAACCAG AAGTTCAAGG 1501 GCAAAGCCAC ACTGACCGCC GACAAGAGCA GCAGCACAGC CTACATGCAG CTGAGCAGCC 1561 TGACCAGCGA AGATAGCGCC GTGTACTACT GCGCCAGAAG CACCTATTAC GGCGGCGACT 1621 GGTACTTCAA CGTGTGGGGA GCTGGCACCA CCGTGACAGT GTCTGCCGCC TCTACAAAGG 1681 GCCCTAGCGT TTTCCCACTG GCTCCCAGCA GCAAGAGCAC ATCTGGTGGA ACAGCCGCTC 1741 TGGGCTGCCT GGTCAAGGAT TACTTTCCCG AGCCTGTGAC CGTGTCCTGG AATAGCGGAG 1801 CACTGACAAG CGGCGTGCAC ACCTTTCCAG CAGTGCTGCA AAGCAGCGGC CTGTACTCTC 1861 TGAGCAGCGT GGTCACAGTG CCTAGCTCTA GCCTGGGCAC CCAGACCTAC ATCTGCAATG 1921 TGAACCACAA GCCTAGCAAC ACCAAGGTGG ACAAGAAGGT GGAACCCAAG AGCTGCGACA 1981 AGACCCACAC CTGTCCTCCA TGTCCTGCTC CAGAACTGCT CGGCGGACCT TCCGTGTTCC 2041 TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG 2101 TGGTGGTGGA TGTGTCTCAC GAGGACCCCG AAGTGAAGTT CAATTGGTAC GTGGACGGCG 2161 TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTACAACAGC ACCTACAGAG 2221 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA 2281 AGGTGTCCAA CAAGGCCCTG CCTGCTCCTA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC 2341 AGCCTAGGGA ACCCCAGGTT TACACACTGC CTCCAAGCAG GGACGAGCTG ACCAAGAATC 2401 AGGTGTCCCT GACCTGCCTC GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG2461 AGAGCAATGG CCAGCCTGAG AACAACTACA AGACAACCCC TCCTGTGCTG GACAGCGACG 2521 GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CAGATGGCAG CAGGGCAACG 2581 TGTTCTCCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAAAGCCTGT 2641 CTCTGAGCCC CGGCAAGCGA AAAAGAAGAT CAGGTTCGGG TGCGCCAGTA AAGCAGACAT 2701 TAAACTTTGA TTTGCTGAAA CTTGCAGGTG ATGTAGAGTC AAATCCAGGT CCAATGGCAA 2761 CAGGGAGCCG AACCTCTCTG CTCCTTGCTT TCGGGCTCCT TTGCCTACCG TGGCTCCAAG 2821 AGGGCTCGGC ACAGATCGTG CTGTCTCAGT CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG 2881 AGAAAGTGAC CATGACCTGT AGAGCCAGCA GCAGCGTGTC CTACATCCAC TGGTTCCAGC 2941 AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA TCTACGCCAC AAGCAATCTG GCCAGCGGCG 3001 TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG GCACCAGCTA CAGCCTGACA ATCAGCAGAG 3061 TGGAAGCCGA GGATGCCGCC ACCTACTACT GTCAGCAGTG GACCAGCAAT CCTCCTACCT 3121 TTGGCGGAGG CACCAAGCTG GAAATCAAGC GGACAGTGGC CGCTCCTAGC GTGTTCATCT 3181 TTCCACCTAG CGACGAGCAG CTGAAGTCTG GCACAGCTAG CGTCGTGTGC CTGCTGAACA 3241 ACTTCTACCC CAGAGAAGCC AAGGTGCAGT GGAAGGTGGA CAACGCCCTG CAGAGCGGCA 3301 ATAGCCAAGA GAGCGTGACC GAGCAGGACA GCAAGGACTC CACCTATAGC CTGAGCAGCA 3361 CACTGACCCT GAGCAAGGCC GACTACGAGA AGCACAAAGT GTACGCCTGC GAAGTGACCC 3421 ACCAGGGCCT TTCTAGCCCT GTGACCAAGA GCTTCAACCG GGGCGAGTGT TGAGaattcg 3481 atatcaagct tatcgataat caacctctgg attacaaaat ttgtgaaaga ttgactggta 3541 ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc 3601 atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt 3661 ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg 3721 ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt 3781 tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct 3841 ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg aaatcatcgt 3901 cctttccttg gctgctcgcc tatgttgcca cctggattct gcgcgggacg tccttctgct 3961 acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc 4021 ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct 4081 ccccgcCTCG AGcatggccc aacttgttta ttgcagctta taatggttac aaataaagca 4141 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 4201 ccaaactcat caatgtatct tatcatgtct ggatctccgg accacgtgcg gaccgagcgg 4261 ccgcaggaac ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact 4321 gaggccgggc gaccaaaggt cgcccgacgc ccgggctttg cccgggcggc ctcagtgagc 4381 gagcgagcgc gcagctgcct gcagg BRV-P-002 (SEQ ID NO: 163) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG CGACGGGTTC AAGAACTTCC CTACTTCTTG 1261 CATTTGGCCT GCTTTGTTTG CCGTGGTTAC AGGAGGGCTC GGCACAGGTT CAGCTGCAAC1321 AGCCTGGCGC CGAACTTGTG AAACCTGGCG CCTCTGTGAA GATGAGCTGC AAGGCCAGCG 1381 GCTACACCTT CACCAGCTAC AACATGCACT GGGTCAAGCA GACCCCTGGC AGAGGCCTGG 1441 AATGGATCGG AGCCATCTAT CCCGGCAACG GCGACACCTC CTACAACCAG AAGTTCAAGG 1501 GCAAAGCCAC ACTGACCGCC GACAAGAGCA GCAGCACAGC CTACATGCAG CTGAGCAGCC 1561 TGACCAGCGA AGATAGCGCC GTGTACTACT GCGCCAGAAG CACCTATTAC GGCGGCGACT 1621 GGTACTTCAA CGTGTGGGGA GCTGGCACCA CCGTGACAGT GTCTGCCGCC TCTACAAAGG 1681 GCCCTAGCGT TTTCCCACTG GCTCCCAGCA GCAAGAGCAC ATCTGGTGGA ACAGCCGCTC 1741 TGGGCTGCCT GGTCAAGGAT TACTTTCCCG AGCCTGTGAC CGTGTCCTGG AATAGCGGAG 1801 CACTGACAAG CGGCGTGCAC ACCTTTCCAG CAGTGCTGCA AAGCAGCGGC CTGTACTCTC 1861 TGAGCAGCGT GGTCACAGTG CCTAGCTCTA GCCTGGGCAC CCAGACCTAC ATCTGCAATG 1921 TGAACCACAA GCCTAGCAAC ACCAAGGTGG ACAAGAAGGT GGAACCCAAG AGCTGCGACA 1981 AGACCCACAC CTGTCCTCCA TGTCCTGCTC CAGAACTGCT CGGCGGACCT TCCGTGTTCC 2041 TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG 2101 TGGTGGTGGA TGTGTCTCAC GAGGACCCCG AAGTGAAGTT CAATTGGTAC GTGGACGGCG 2161 TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTACAACAGC ACCTACAGAG 2221 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA 2281 AGGTGTCCAA CAAGGCCCTG CCTGCTCCTA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC 2341 AGCCTAGGGA ACCCCAGGTT TACACACTGC CTCCAAGCAG GGACGAGCTG ACCAAGAATC 2401 AGGTGTCCCT GACCTGCCTC GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG 2461 AGAGCAATGG CCAGCCTGAG AACAACTACA AGACAACCCC TCCTGTGCTG GACAGCGACG 2521 GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CAGATGGCAG CAGGGCAACG 2581 TGTTCTCCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAAAGCCTGT 2641 CTCTGAGCCC CGGCAAGCGA AAAAGAAGAT CAGGTTCGGG TGCGCCAGTA AAGCAGACAT 2701 TAAACTTTGA TTTGCTGAAA CTTGCAGGTG ATGTAGAGTC AAATCCAGGT CCAATGGCAA 2761 CAGGGAGCCG AACCTCTCTG CTCCTTGCTT TCGGGCTCCT TTGCCTACCG TGGCTCCAAG 2821 AGGGCTCGGC ACAGATCGTG CTGTCTCAGT CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG 2881 AGAAAGTGAC CATGACCTGT AGAGCCAGCA GCAGCGTGTC CTACATCCAC TGGTTCCAGC 2941 AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA TCTACGCCAC AAGCAATCTG GCCAGCGGCG 3001 TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG GCACCAGCTA CAGCCTGACA ATCAGCAGAG 3061 TGGAAGCCGA GGATGCCGCC ACCTACTACT GTCAGCAGTG GACCAGCAAT CCTCCTACCT 3121 TTGGCGGAGG CACCAAGCTG GAAATCAAGC GGACAGTGGC CGCTCCTAGC GTGTTCATCT 3181 TTCCACCTAG CGACGAGCAG CTGAAGTCTG GCACAGCTAG CGTCGTGTGC CTGCTGAACA 3241 ACTTCTACCC CAGAGAAGCC AAGGTGCAGT GGAAGGTGGA CAACGCCCTG CAGAGCGGCA 3301 ATAGCCAAGA GAGCGTGACC GAGCAGGACA GCAAGGACTC CACCTATAGC CTGAGCAGCA 3361 CACTGACCCT GAGCAAGGCC GACTACGAGA AGCACAAAGT GTACGCCTGC GAAGTGACCC 3421 ACCAGGGCCT TTCTAGCCCT GTGACCAAGA GCTTCAACCG GGGCGAGTGT TGAGaattcT 3481 GAGTCCATAA AGTAGGAAAC ACTACATGAG TCCATAAAGT AGGAAACACT ACATGAGTCC 3541 ATAAAGTAGG AAACACTACA TGAGTCCATA AAGTAGGAAA CACTACATGA Gaagcttatc 3601 gataatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 3661 gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 3721 cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 3781 ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 3841 actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 3901 cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 3961 ctgttgggca ctgacaattc cgtggtgttg tcggggaaat catcgtcctt tccttggctg 4021 ctcgcctatg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 4081 ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 4141 cttcgccttc gccctcagac gagtcggatc tccctttggg ccgcctcccc gcCTCGAGca 4201 tggcccaact tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat 4261 ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat 4321 gtatcttatc atgtctggat ctccggacca cgtgcggacc gagcggccgc aggaacccct 4381 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc 4441 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag 4501 ctgcctgcag g BRV-P-003 (SEQ ID NO: 164) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG CGACGGGTTC AAGAACTTCC CTACTTCTTG 1261 CATTTGGCCT GCTTTGTTTG CCGTGGTTAC AGGAGGGCTC GGCACAGGTT CAGCTGCAAC 1321 AGCCTGGCGC CGAACTTGTG AAACCTGGCG CCTCTGTGAA GATGAGCTGC AAGGCCAGCG 1381 GCTACACCTT CACCAGCTAC AACATGCACT GGGTCAAGCA GACCCCTGGC AGAGGCCTGG 1441 AATGGATCGG AGCCATCTAT CCCGGCAACG GCGACACCTC CTACAACCAG AAGTTCAAGG 1501 GCAAAGCCAC ACTGACCGCC GACAAGAGCA GCAGCACAGC CTACATGCAG CTGAGCAGCC 1561 TGACCAGCGA AGATAGCGCC GTGTACTACT GCGCCAGAAG CACCTATTAC GGCGGCGACT 1621 GGTACTTCAA CGTGTGGGGA GCTGGCACCA CCGTGACAGT GTCTGCCGCC TCTACAAAGG 1681 GCCCTAGCGT TTTCCCACTG GCTCCCAGCA GCAAGAGCAC ATCTGGTGGA ACAGCCGCTC 1741 TGGGCTGCCT GGTCAAGGAT TACTTTCCCG AGCCTGTGAC CGTGTCCTGG AATAGCGGAG 1801 CACTGACAAG CGGCGTGCAC ACCTTTCCAG CAGTGCTGCA AAGCAGCGGC CTGTACTCTC 1861 TGAGCAGCGT GGTCACAGTG CCTAGCTCTA GCCTGGGCAC CCAGACCTAC ATCTGCAATG 1921 TGAACCACAA GCCTAGCAAC ACCAAGGTGG ACAAGAAGGT GGAACCCAAG AGCTGCGACA 1981 AGACCCACAC CTGTCCTCCA TGTCCTGCTC CAGAACTGCT CGGCGGACCT TCCGTGTTCC 2041 TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG 2101 TGGTGGTGGA TGTGTCTCAC GAGGACCCCG AAGTGAAGTT CAATTGGTAC GTGGACGGCG 2161 TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTACAACAGC ACCTACAGAG 2221 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA 2281 AGGTGTCCAA CAAGGCCCTG CCTGCTCCTA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC 2341 AGCCTAGGGA ACCCCAGGTT TACACACTGC CTCCAAGCAG GGACGAGCTG ACCAAGAATC 2401 AGGTGTCCCT GACCTGCCTC GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG 2461 AGAGCAATGG CCAGCCTGAG AACAACTACA AGACAACCCC TCCTGTGCTG GACAGCGACG 2521 GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CAGATGGCAG CAGGGCAACG 2581 TGTTCTCCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAAAGCCTGT 2641 CTCTGAGCCC CGGCAAGCGA AAAAGAAGAT CAGGTTCGGG TGCGCCAGTA AAGCAGACAT 2701 TAAACTTTGA TTTGCTGAAA CTTGCAGGTG ATGTAGAGTC AAATCCAGGT CCAATGGCAA 2761 CAGGGAGCCG AACCTCTCTG CTCCTTGCTT TCGGGCTCCT TTGCCTACCG TGGCTCCAAG 2821 AGGGCTCGGC ACAGATCGTG CTGTCTCAGT CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG 2881 AGAAAGTGAC CATGACCTGT AGAGCCAGCA GCAGCGTGTC CTACATCCAC TGGTTCCAGC 2941 AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA TCTACGCCAC AAGCAATCTG GCCAGCGGCG 3001 TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG GCACCAGCTA CAGCCTGACA ATCAGCAGAG 3061 TGGAAGCCGA GGATGCCGCC ACCTACTACT GTCAGCAGTG GACCAGCAAT CCTCCTACCT 3121 TTGGCGGAGG CACCAAGCTG GAAATCAAGC GGACAGTGGC CGCTCCTAGC GTGTTCATCT 3181 TTCCACCTAG CGACGAGCAG CTGAAGTCTG GCACAGCTAG CGTCGTGTGC CTGCTGAACA 3241 ACTTCTACCC CAGAGAAGCC AAGGTGCAGT GGAAGGTGGA CAACGCCCTG CAGAGCGGCA 3301 ATAGCCAAGA GAGCGTGACC GAGCAGGACA GCAAGGACTC CACCTATAGC CTGAGCAGCA 3361 CACTGACCCT GAGCAAGGCC GACTACGAGA AGCACAAAGT GTACGCCTGC GAAGTGACCC 3421 ACCAGGGCCT TTCTAGCCCT GTGACCAAGA GCTTCAACCG GGGCGAGTGT TGAGaattcT3481 GAGCAAACAC CATTGTCACA CTCCATGAGC AAACACCATT GTCACACTCC ATGAGCAAAC 3541 ACCATTGTCA CACTCCATGA GCAAACACCA TTGTCACACT CCATGAGCAA ACACCATTGT 3601 CACACTCCAT GAGaagctta tcgataatca acctctggat tacaaaattt gtgaaagatt 3661 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 3721 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 3781 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 3841 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 3901 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 3961 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 4021 atcatcgtcc tttccttggc tgctcgccta tgttgccacc tggattctgc gcgggacgtc 4081 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 4141 ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 4201 ggccgcctcc ccgcCTCGAG catggcccaa cttgtttatt gcagcttata atggttacaa 4261 ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg 4321 tggtttgtcc aaactcatca atgtatctta tcatgtctgg atctccggac cacgtgcgga 4381 ccgagcggcc gcaggaaccc ctagtgatgg agttggccac tccctctctg cgcgctcgct 4441 cgctcactga ggccgggcga ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct 4501 cagtgagcga gcgagcgcgc agctgcctgc agg BRV-P-004 (SEQ ID NO: 165) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG CGACGGGTTC AAGAACTTCC CTACTTCTTG 1261 CATTTGGCCT GCTTTGTTTG CCGTGGTTAC AGGAGGGCTC GGCACAGGTT CAGCTGCAAC 1321 AGCCTGGCGC CGAACTTGTG AAACCTGGCG CCTCTGTGAA GATGAGCTGC AAGGCCAGCG 1381 GCTACACCTT CACCAGCTAC AACATGCACT GGGTCAAGCA GACCCCTGGC AGAGGCCTGG 1441 AATGGATCGG AGCCATCTAT CCCGGCAACG GCGACACCTC CTACAACCAG AAGTTCAAGG 1501 GCAAAGCCAC ACTGACCGCC GACAAGAGCA GCAGCACAGC CTACATGCAG CTGAGCAGCC 1561 TGACCAGCGA AGATAGCGCC GTGTACTACT GCGCCAGAAG CACCTATTAC GGCGGCGACT 1621 GGTACTTCAA CGTGTGGGGA GCTGGCACCA CCGTGACAGT GTCTGCCGCC TCTACAAAGG 1681 GCCCTAGCGT TTTCCCACTG GCTCCCAGCA GCAAGAGCAC ATCTGGTGGA ACAGCCGCTC 1741 TGGGCTGCCT GGTCAAGGAT TACTTTCCCG AGCCTGTGAC CGTGTCCTGG AATAGCGGAG 1801 CACTGACAAG CGGCGTGCAC ACCTTTCCAG CAGTGCTGCA AAGCAGCGGC CTGTACTCTC 1861 TGAGCAGCGT GGTCACAGTG CCTAGCTCTA GCCTGGGCAC CCAGACCTAC ATCTGCAATG 1921 TGAACCACAA GCCTAGCAAC ACCAAGGTGG ACAAGAAGGT GGAACCCAAG AGCTGCGACA 1981 AGACCCACAC CTGTCCTCCA TGTCCTGCTC CAGAACTGCT CGGCGGACCT TCCGTGTTCC 2041 TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG 2101 TGGTGGTGGA TGTGTCTCAC GAGGACCCCG AAGTGAAGTT CAATTGGTAC GTGGACGGCG 2161 TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTACAACAGC ACCTACAGAG2221 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA 2281 AGGTGTCCAA CAAGGCCCTG CCTGCTCCTA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC 2341 AGCCTAGGGA ACCCCAGGTT TACACACTGC CTCCAAGCAG GGACGAGCTG ACCAAGAATC 2401 AGGTGTCCCT GACCTGCCTC GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG 2461 AGAGCAATGG CCAGCCTGAG AACAACTACA AGACAACCCC TCCTGTGCTG GACAGCGACG 2521 GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CAGATGGCAG CAGGGCAACG 2581 TGTTCTCCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAAAGCCTGT 2641 CTCTGAGCCC CGGCAAGCGA AAAAGAAGAT CAGGTTCGGG TGCGCCAGTA AAGCAGACAT 2701 TAAACTTTGA TTTGCTGAAA CTTGCAGGTG ATGTAGAGTC AAATCCAGGT CCAATGGCAA 2761 CAGGGAGCCG AACCTCTCTG CTCCTTGCTT TCGGGCTCCT TTGCCTACCG TGGCTCCAAG 2821 AGGGCTCGGC ACAGATCGTG CTGTCTCAGT CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG 2881 AGAAAGTGAC CATGACCTGT AGAGCCAGCA GCAGCGTGTC CTACATCCAC TGGTTCCAGC 2941 AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA TCTACGCCAC AAGCAATCTG GCCAGCGGCG 3001 TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG GCACCAGCTA CAGCCTGACA ATCAGCAGAG 3061 TGGAAGCCGA GGATGCCGCC ACCTACTACT GTCAGCAGTG GACCAGCAAT CCTCCTACCT 3121 TTGGCGGAGG CACCAAGCTG GAAATCAAGC GGACAGTGGC CGCTCCTAGC GTGTTCATCT 3181 TTCCACCTAG CGACGAGCAG CTGAAGTCTG GCACAGCTAG CGTCGTGTGC CTGCTGAACA 3241 ACTTCTACCC CAGAGAAGCC AAGGTGCAGT GGAAGGTGGA CAACGCCCTG CAGAGCGGCA 3301 ATAGCCAAGA GAGCGTGACC GAGCAGGACA GCAAGGACTC CACCTATAGC CTGAGCAGCA 3361 CACTGACCCT GAGCAAGGCC GACTACGAGA AGCACAAAGT GTACGCCTGC GAAGTGACCC 3421 ACCAGGGCCT TTCTAGCCCT GTGACCAAGA GCTTCAACCG GGGCGAGTGT TGAGaattcT 3481 GAGTCCATAA AGTAGGAAAC ACTACATGAG TCCATAAAGT AGGAAACACT ACATGAGTCC 3541 ATAAAGTAGG AAACACTACA TGAGTCCATA AAGTAGGAAA CACTACATGA GgatatcTGA 3601 GCAAACACCA TTGTCACACT CCATGAGCAA ACACCATTGT CACACTCCAT GAGCAAACAC 3661 CATTGTCACA CTCCATGAGC AAACACCATT GTCACACTCC ATGAGCAAAC ACCATTGTCA 3721 CACTCCATGA Gaagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga 3781 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 3841 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 3901 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 3961 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 4021 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 4081 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 4141 catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct 4201 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 4261 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 4321 ccgcctcccc gcCTCGAGca tggcccaact tgtttattgc agcttataat ggttacaaat 4381 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 4441 gtttgtccaa actcatcaat gtatcttatc atgtctggat ctccggacca cgtgcggacc 4501 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4561 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4621 gtgagcgagc gagcgcgcag ctgcctgcag g BRV-P-005 (SEQ ID NO: 166) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG CGACGGGTTC AAGAACTTCC CTACTTCTTG 1261 CATTTGGCCT GCTTTGTTTG CCGTGGTTAC AGGAGGGCTC GGCACAGGTT CAGCTGCAAC 1321 AGCCTGGCGC CGAACTTGTG AAACCTGGCG CCTCTGTGAA GATGAGCTGC AAGGCCAGCG 1381 GCTACACCTT CACCAGCTAC AACATGCACT GGGTCAAGCA GACCCCTGGC AGAGGCCTGG 1441 AATGGATCGG AGCCATCTAT CCCGGCAACG GCGACACCTC CTACAACCAG AAGTTCAAGG 1501 GCAAAGCCAC ACTGACCGCC GACAAGAGCA GCAGCACAGC CTACATGCAG CTGAGCAGCC 1561 TGACCAGCGA AGATAGCGCC GTGTACTACT GCGCCAGAAG CACCTATTAC GGCGGCGACT 1621 GGTACTTCAA CGTGTGGGGA GCTGGCACCA CCGTGACAGT GTCTGCCGCC TCTACAAAGG 1681 GCCCTAGCGT TTTCCCACTG GCTCCCAGCA GCAAGAGCAC ATCTGGTGGA ACAGCCGCTC 1741 TGGGCTGCCT GGTCAAGGAT TACTTTCCCG AGCCTGTGAC CGTGTCCTGG AATAGCGGAG 1801 CACTGACAAG CGGCGTGCAC ACCTTTCCAG CAGTGCTGCA AAGCAGCGGC CTGTACTCTC 1861 TGAGCAGCGT GGTCACAGTG CCTAGCTCTA GCCTGGGCAC CCAGACCTAC ATCTGCAATG 1921 TGAACCACAA GCCTAGCAAC ACCAAGGTGG ACAAGAAGGT GGAACCCAAG AGCTGCGACA 1981 AGACCCACAC CTGTCCTCCA TGTCCTGCTC CAGAACTGCT CGGCGGACCT TCCGTGTTCC 2041 TGTTTCCTCC AAAGCCTAAG GACACCCTGA TGATCAGCAG AACCCCTGAA GTGACCTGCG 2101 TGGTGGTGGA TGTGTCTCAC GAGGACCCCG AAGTGAAGTT CAATTGGTAC GTGGACGGCG 2161 TGGAAGTGCA CAACGCCAAG ACCAAGCCTA GAGAGGAACA GTACAACAGC ACCTACAGAG 2221 TGGTGTCCGT GCTGACCGTG CTGCACCAGG ATTGGCTGAA CGGCAAAGAG TACAAGTGCA 2281 AGGTGTCCAA CAAGGCCCTG CCTGCTCCTA TCGAGAAAAC CATCAGCAAG GCCAAGGGCC 2341 AGCCTAGGGA ACCCCAGGTT TACACACTGC CTCCAAGCAG GGACGAGCTG ACCAAGAATC 2401 AGGTGTCCCT GACCTGCCTC GTGAAGGGCT TCTACCCTTC CGATATCGCC GTGGAATGGG 2461 AGAGCAATGG CCAGCCTGAG AACAACTACA AGACAACCCC TCCTGTGCTG GACAGCGACG 2521 GCTCATTCTT CCTGTACAGC AAGCTGACAG TGGACAAGTC CAGATGGCAG CAGGGCAACG 2581 TGTTCTCCTG CAGCGTGATG CACGAGGCCC TGCACAACCA CTACACCCAG AAAAGCCTGT 2641 CTCTGAGCCC CGGCAAGCGA AAAAGAAGAT CAGGTTCGGG TGCGCCAGTA AAGCAGACAT 2701 TAAACTTTGA TTTGCTGAAA CTTGCAGGTG ATGTAGAGTC AAATCCAGGT CCAATGGCAA 2761 CAGGGAGCCG AACCTCTCTG CTCCTTGCTT TCGGGCTCCT TTGCCTACCG TGGCTCCAAG 2821 AGGGCTCGGC ACAGATCGTG CTGTCTCAGT CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG 2881 AGAAAGTGAC CATGACCTGT AGAGCCAGCA GCAGCGTGTC CTACATCCAC TGGTTCCAGC 2941 AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA TCTACGCCAC AAGCAATCTG GCCAGCGGCG 3001 TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG GCACCAGCTA CAGCCTGACA ATCAGCAGAG 3061 TGGAAGCCGA GGATGCCGCC ACCTACTACT GTCAGCAGTG GACCAGCAAT CCTCCTACCT 3121 TTGGCGGAGG CACCAAGCTG GAAATCAAGC GGACAGTGGC CGCTCCTAGC GTGTTCATCT 3181 TTCCACCTAG CGACGAGCAG CTGAAGTCTG GCACAGCTAG CGTCGTGTGC CTGCTGAACA 3241 ACTTCTACCC CAGAGAAGCC AAGGTGCAGT GGAAGGTGGA CAACGCCCTG CAGAGCGGCA 3301 ATAGCCAAGA GAGCGTGACC GAGCAGGACA GCAAGGACTC CACCTATAGC CTGAGCAGCA 3361 CACTGACCCT GAGCAAGGCC GACTACGAGA AGCACAAAGT GTACGCCTGC GAAGTGACCC 3421 ACCAGGGCCT TTCTAGCCCT GTGACCAAGA GCTTCAACCG GGGCGAGTGT TGAGaattcg 3481 atatcaagct TCGAGcatgg cccaacttgt ttattgcagc ttataatggt tacaaataaa 3541 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 3601 tgtccaaact catcaatgta tcttatcatg tctggatctc cggaccacgt gcggaccgag 3661 cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3721 actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 3781 agcgagcgag cgcgcagctg cctgcagg BRV-P-006 (SEQ ID NO: 167) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG ACATGAGAGT GCCCGCTCAA CTGCTGGGAC 1261 TGCTGCTGCT TTGGCTGAGA GGTGCTAGAT GCCAGGTTCA GCTGCAACAG CCTGGCGCCG 1321 AACTTGTGAA ACCTGGCGCC TCTGTGAAGA TGAGCTGCAA GGCCAGCGGC TACACCTTCA 1381 CCAGCTACAA CATGCACTGG GTCAAGCAGA CCCCTGGCAG AGGCCTGGAA TGGATCGGAG 1441 CCATCTATCC CGGCAACGGC GACACCTCCT ACAACCAGAA GTTCAAGGGC AAAGCCACAC 1501 TGACCGCCGA CAAGAGCAGC AGCACAGCCT ACATGCAGCT GAGCAGCCTG ACCAGCGAAG 1561 ATAGCGCCGT GTACTACTGC GCCAGAAGCA CCTATTACGG CGGCGACTGG TACTTCAACG 1621 TGTGGGGAGC TGGCACCACC GTGACAGTGT CTGCCGCCTC TACAAAGGGC CCTAGCGTTT 1681 TCCCACTGGC TCCCAGCAGC AAGAGCACAT CTGGTGGAAC AGCCGCTCTG GGCTGCCTGG 1741 TCAAGGATTA CTTTCCCGAG CCTGTGACCG TGTCCTGGAA TAGCGGAGCA CTGACAAGCG 1801 GCGTGCACAC CTTTCCAGCA GTGCTGCAAA GCAGCGGCCT GTACTCTCTG AGCAGCGTGG 1861 TCACAGTGCC TAGCTCTAGC CTGGGCACCC AGACCTACAT CTGCAATGTG AACCACAAGC 1921 CTAGCAACAC CAAGGTGGAC AAGAAGGTGG AACCCAAGAG CTGCGACAAG ACCCACACCT 1981 GTCCTCCATG TCCTGCTCCA GAACTGCTCG GCGGACCTTC CGTGTTCCTG TTTCCTCCAA 2041 AGCCTAAGGA CACCCTGATG ATCAGCAGAA CCCCTGAAGT GACCTGCGTG GTGGTGGATG 2101 TGTCTCACGA GGACCCCGAA GTGAAGTTCA ATTGGTACGT GGACGGCGTG GAAGTGCACA 2161 ACGCCAAGAC CAAGCCTAGA GAGGAACAGT ACAACAGCAC CTACAGAGTG GTGTCCGTGC 2221 TGACCGTGCT GCACCAGGAT TGGCTGAACG GCAAAGAGTA CAAGTGCAAG GTGTCCAACA 2281 AGGCCCTGCC TGCTCCTATC GAGAAAACCA TCAGCAAGGC CAAGGGCCAG CCTAGGGAAC 2341 CCCAGGTTTA CACACTGCCT CCAAGCAGGG ACGAGCTGAC CAAGAATCAG GTGTCCCTGA 2401 CCTGCCTCGT GAAGGGCTTC TACCCTTCCG ATATCGCCGT GGAATGGGAG AGCAATGGCC 2461 AGCCTGAGAA CAACTACAAG ACAACCCCTC CTGTGCTGGA CAGCGACGGC TCATTCTTCC 2521 TGTACAGCAA GCTGACAGTG GACAAGTCCA GATGGCAGCA GGGCAACGTG TTCTCCTGCA 2581 GCGTGATGCA CGAGGCCCTG CACAACCACT ACACCCAGAA AAGCCTGTCT CTGAGCCCCG 2641 GCAAGCGAAA AAGAAGATCA GGTTCGGGTG CGCCAGTAAA GCAGACATTA AACTTTGATT 2701 TGCTGAAACT TGCAGGTGAT GTAGAGTCAA ATCCAGGTCC AATGTACCGG ATGCAGCTGC 2761 TGAGCTGTAT CGCCCTGTCT CTGGCCCTGG TCACCAACTC TCAGATCGTG CTGTCTCAGT 2821 CTCCTGCCAT CCTGTCTGCT AGCCCTGGCG AGAAAGTGAC CATGACCTGT AGAGCCAGCA 2881 GCAGCGTGTC CTACATCCAC TGGTTCCAGC AGAAGCCTGG CAGCAGCCCT AAGCCTTGGA 2941 TCTACGCCAC AAGCAATCTG GCCAGCGGCG TGCCAGTCAG ATTTTCTGGC TCTGGCAGCG 3001 GCACCAGCTA CAGCCTGACA ATCAGCAGAG TGGAAGCCGA GGATGCCGCC ACCTACTACT 3061 GTCAGCAGTG GACCAGCAAT CCTCCTACCT TTGGCGGAGG CACCAAGCTG GAAATCAAGC 3121 GGACAGTGGC CGCTCCTAGC GTGTTCATCT TTCCACCTAG CGACGAGCAG CTGAAGTCTG 3181 GCACAGCTAG CGTCGTGTGC CTGCTGAACA ACTTCTACCC CAGAGAAGCC AAGGTGCAGT 3241 GGAAGGTGGA CAACGCCCTG CAGAGCGGCA ATAGCCAAGA GAGCGTGACC GAGCAGGACA 3301 GCAAGGACTC CACCTATAGC CTGAGCAGCA CACTGACCCT GAGCAAGGCC GACTACGAGA 3361 AGCACAAAGT GTACGCCTGC GAAGTGACCC ACCAGGGCCT TTCTAGCCCT GTGACCAAGA 3421 GCTTCAACCG GGGCGAGTGT TGAGaattcg atatcaagct tatcgataat caacctctgg 3481 attacaaaat ttgtgaaaga ttgactggta ttcttaacta tgttgctcct tttacgctat 3541 gtggatacgc tgctttaatg cctttgtatc atgctattgc ttcccgtatg gctttcattt 3601 tctcctcctt gtataaatcc tggttgctgt ctctttatga ggagttgtgg cccgttgtca 3661 ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt tggggcattg3721 ccaccacctg tcagctcctt tccgggactt tcgctttccc cctccctatt gccacggcgg 3781 aactcatcgc cgcctgcctt gcccgctgct ggacaggggc tcggctgttg ggcactgaca 3841 attccgtggt gttgtcgggg aaatcatcgt cctttccttg gctgctcgcc tatgttgcca 3901 cctggattct gcgcgggacg tccttctgct acgtcccttc ggccctcaat ccagcggacc 3961 ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc 4021 agacgagtcg gatctccctt tgggccgcct ccccgcCTCG AGcatggccc aacttgttta 4081 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 4141 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 4201 ggatctccgg accacgtgcg gaccgagcgg ccgcaggaac ccctagtgat ggagttggcc 4261 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc 4321 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcagg BRV-P-007 (SEQ ID NO: 168) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGG AGTTTGGGCT GAGCTGGGTT TTCCTCGTTG 1261 CTCTTTTTAG AGGTGTCCAG TGTCAGGTTC AGCTGCAACA GCCTGGCGCC GAACTTGTGA 1321 AACCTGGCGC CTCTGTGAAG ATGAGCTGCA AGGCCAGCGG CTACACCTTC ACCAGCTACA 1381 ACATGCACTG GGTCAAGCAG ACCCCTGGCA GAGGCCTGGA ATGGATCGGA GCCATCTATC 1441 CCGGCAACGG CGACACCTCC TACAACCAGA AGTTCAAGGG CAAAGCCACA CTGACCGCCG 1501 ACAAGAGCAG CAGCACAGCC TACATGCAGC TGAGCAGCCT GACCAGCGAA GATAGCGCCG 1561 TGTACTACTG CGCCAGAAGC ACCTATTACG GCGGCGACTG GTACTTCAAC GTGTGGGGAG 1621 CTGGCACCAC CGTGACAGTG TCTGCCGCCT CTACAAAGGG CCCTAGCGTT TTCCCACTGG 1681 CTCCCAGCAG CAAGAGCACA TCTGGTGGAA CAGCCGCTCT GGGCTGCCTG GTCAAGGATT 1741 ACTTTCCCGA GCCTGTGACC GTGTCCTGGA ATAGCGGAGC ACTGACAAGC GGCGTGCACA 1801 CCTTTCCAGC AGTGCTGCAA AGCAGCGGCC TGTACTCTCT GAGCAGCGTG GTCACAGTGC 1861 CTAGCTCTAG CCTGGGCACC CAGACCTACA TCTGCAATGT GAACCACAAG CCTAGCAACA 1921 CCAAGGTGGA CAAGAAGGTG GAACCCAAGA GCTGCGACAA GACCCACACC TGTCCTCCAT 1981 GTCCTGCTCC AGAACTGCTC GGCGGACCTT CCGTGTTCCT GTTTCCTCCA AAGCCTAAGG 2041 ACACCCTGAT GATCAGCAGA ACCCCTGAAG TGACCTGCGT GGTGGTGGAT GTGTCTCACG 2101 AGGACCCCGA AGTGAAGTTC AATTGGTACG TGGACGGCGT GGAAGTGCAC AACGCCAAGA 2161 CCAAGCCTAG AGAGGAACAG TACAACAGCA CCTACAGAGT GGTGTCCGTG CTGACCGTGC 2221 TGCACCAGGA TTGGCTGAAC GGCAAAGAGT ACAAGTGCAA GGTGTCCAAC AAGGCCCTGC 2281 CTGCTCCTAT CGAGAAAACC ATCAGCAAGG CCAAGGGCCA GCCTAGGGAA CCCCAGGTTT 2341 ACACACTGCC TCCAAGCAGG GACGAGCTGA CCAAGAATCA GGTGTCCCTG ACCTGCCTCG 2401 TGAAGGGCTT CTACCCTTCC GATATCGCCG TGGAATGGGA GAGCAATGGC CAGCCTGAGA 2461 ACAACTACAA GACAACCCCT CCTGTGCTGG ACAGCGACGG CTCATTCTTC CTGTACAGCA 2521 AGCTGACAGT GGACAAGTCC AGATGGCAGC AGGGCAACGT GTTCTCCTGC AGCGTGATGC 2581 ACGAGGCCCT GCACAACCAC TACACCCAGA AAAGCCTGTC TCTGAGCCCC GGCAAGCGAA2641 AAAGAAGATC AGGTTCGGGT GCGCCAGTAA AGCAGACATT AAACTTTGAT TTGCTGAAAC 2701 TTGCAGGTGA TGTAGAGTCA AATCCAGGTC CAATGAAATA CCTATTGCCT ACGGCAGCCG 2761 CTGGATTGTT ATTACTCGCG GCCCAGCCGG CCATGGCCCA GATCGTGCTG TCTCAGTCTC 2821 CTGCCATCCT GTCTGCTAGC CCTGGCGAGA AAGTGACCAT GACCTGTAGA GCCAGCAGCA 2881 GCGTGTCCTA CATCCACTGG TTCCAGCAGA AGCCTGGCAG CAGCCCTAAG CCTTGGATCT 2941 ACGCCACAAG CAATCTGGCC AGCGGCGTGC CAGTCAGATT TTCTGGCTCT GGCAGCGGCA 3001 CCAGCTACAG CCTGACAATC AGCAGAGTGG AAGCCGAGGA TGCCGCCACC TACTACTGTC 3061 AGCAGTGGAC CAGCAATCCT CCTACCTTTG GCGGAGGCAC CAAGCTGGAA ATCAAGCGGA 3121 CAGTGGCCGC TCCTAGCGTG TTCATCTTTC CACCTAGCGA CGAGCAGCTG AAGTCTGGCA 3181 CAGCTAGCGT CGTGTGCCTG CTGAACAACT TCTACCCCAG AGAAGCCAAG GTGCAGTGGA 3241 AGGTGGACAA CGCCCTGCAG AGCGGCAATA GCCAAGAGAG CGTGACCGAG CAGGACAGCA 3301 AGGACTCCAC CTATAGCCTG AGCAGCACAC TGACCCTGAG CAAGGCCGAC TACGAGAAGC 3361 ACAAAGTGTA CGCCTGCGAA GTGACCCACC AGGGCCTTTC TAGCCCTGTG ACCAAGAGCT 3421 TCAACCGGGG CGAGTGTTGA Gaattcgata tcaagcttat cgataatcaa cctctggatt 3481 acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 3541 gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 3601 cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 3661 aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 3721 ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 3781 tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 3841 ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct gctcgcctat gttgccacct 3901 ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 3961 cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 4021 cgagtcggat ctccctttgg gccgcctccc cgcCTCGAGc atggcccaac ttgtttattg 4081 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 4141 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga 4201 tctccggacc acgtgcggac cgagcggccg caggaacccc tagtgatgga gttggccact 4261 ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg 4321 ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca gg BRV-P-008 (SEQ ID NO: 169) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagttattaa tagtaatcaa 181 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa 241 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 301 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggac tatttacggt 361 aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 421 tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 481 ctacttggca gtacatctac gtattagtca tcgctattac catggtcgag gtgagcccca 541 cgttctgctt cactctcccc atctcccccc cctccccacc cccaattttg tatttattta 601 ttttttaatt attttgtgca gcgatggggg cggggggggg gggggggcgc gcgccaggcg 661 gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg gcagccaatc 721 agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg cggccctata 781 aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc 841 tccgccgccg cctcgcgccg cccgccccgg ctctgactga ccgcgttact cccacaggtg 901 agcgggcggg acggcccttc tcctccgggc tgtaattagc gcttggttta atgacggctt 961 gtttcttttc tgtggctgcg tgaaagcctt gaggggctcc gggagggccc tttgtgcggg 1021 gggagcggct cggggctgtc cgcgggggga cggctgcctt cgggggggac ggggcagggc 1081 ggggttcggc ttctggcgtg tgaccggcgg ctctagagcc tctgctaacc atgttcatgc 1141 cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt 1201 tggcaaagaa ttggatcccc gccaccATGT GGACCCTGGT GAGCTGGGTG GCCCTGACCG 1261 CCGGCCTGGT GGCCGGCCAG GTTCAGCTGC AACAGCCTGG CGCCGAACTT GTGAAACCTG 1321 GCGCCTCTGT GAAGATGAGC TGCAAGGCCA GCGGCTACAC CTTCACCAGC TACAACATGC 1381 ACTGGGTCAA GCAGACCCCT GGCAGAGGCC TGGAATGGAT CGGAGCCATC TATCCCGGCA 1441 ACGGCGACAC CTCCTACAAC CAGAAGTTCA AGGGCAAAGC CACACTGACC GCCGACAAGA 1501 GCAGCAGCAC AGCCTACATG CAGCTGAGCA GCCTGACCAG CGAAGATAGC GCCGTGTACT1561 ACTGCGCCAG AAGCACCTAT TACGGCGGCG ACTGGTACTT CAACGTGTGG GGAGCTGGCA 1621 CCACCGTGAC AGTGTCTGCC GCCTCTACAA AGGGCCCTAG CGTTTTCCCA CTGGCTCCCA 1681 GCAGCAAGAG CACATCTGGT GGAACAGCCG CTCTGGGCTG CCTGGTCAAG GATTACTTTC 1741 CCGAGCCTGT GACCGTGTCC TGGAATAGCG GAGCACTGAC AAGCGGCGTG CACACCTTTC 1801 CAGCAGTGCT GCAAAGCAGC GGCCTGTACT CTCTGAGCAG CGTGGTCACA GTGCCTAGCT 1861 CTAGCCTGGG CACCCAGACC TACATCTGCA ATGTGAACCA CAAGCCTAGC AACACCAAGG 1921 TGGACAAGAA GGTGGAACCC AAGAGCTGCG ACAAGACCCA CACCTGTCCT CCATGTCCTG 1981 CTCCAGAACT GCTCGGCGGA CCTTCCGTGT TCCTGTTTCC TCCAAAGCCT AAGGACACCC 2041 TGATGATCAG CAGAACCCCT GAAGTGACCT GCGTGGTGGT GGATGTGTCT CACGAGGACC 2101 CCGAAGTGAA GTTCAATTGG TACGTGGACG GCGTGGAAGT GCACAACGCC AAGACCAAGC 2161 CTAGAGAGGA ACAGTACAAC AGCACCTACA GAGTGGTGTC CGTGCTGACC GTGCTGCACC 2221 AGGATTGGCT GAACGGCAAA GAGTACAAGT GCAAGGTGTC CAACAAGGCC CTGCCTGCTC 2281 CTATCGAGAA AACCATCAGC AAGGCCAAGG GCCAGCCTAG GGAACCCCAG GTTTACACAC 2341 TGCCTCCAAG CAGGGACGAG CTGACCAAGA ATCAGGTGTC CCTGACCTGC CTCGTGAAGG 2401 GCTTCTACCC TTCCGATATC GCCGTGGAAT GGGAGAGCAA TGGCCAGCCT GAGAACAACT 2461 ACAAGACAAC CCCTCCTGTG CTGGACAGCG ACGGCTCATT CTTCCTGTAC AGCAAGCTGA 2521 CAGTGGACAA GTCCAGATGG CAGCAGGGCA ACGTGTTCTC CTGCAGCGTG ATGCACGAGG 2581 CCCTGCACAA CCACTACACC CAGAAAAGCC TGTCTCTGAG CCCCGGCAAG CGAAAAAGAA 2641 GATCAGGTTC GGGTGCGCCA GTAAAGCAGA CATTAAACTT TGATTTGCTG AAACTTGCAG 2701 GTGATGTAGA GTCAAATCCA GGTCCAATGT GGACCCTGGT GTCTTGGGTT GCCCTGACAG 2761 CTGGACTGGT GGCCGGACAG ATTGTGCTGT CTCAGTCTCC TGCCATCCTG AGCGCTAGCC 2821 CTGGCGAGAA AGTGACCATG ACCTGTAGAG CCAGCAGCAG CGTGTCCTAC ATCCACTGGT 2881 TCCAGCAGAA GCCTGGCAGC AGCCCTAAGC CTTGGATCTA CGCCACAAGC AATCTGGCCA 2941 GCGGCGTGCC AGTCAGATTT TCTGGCTCTG GCAGCGGCAC CAGCTACAGC CTGACAATCA 3001 GCAGAGTGGA AGCCGAGGAT GCCGCCACCT ACTACTGTCA GCAGTGGACC AGCAATCCTC 3061 CTACCTTTGG CGGAGGCACC AAGCTGGAAA TCAAGCGGAC AGTGGCCGCT CCTAGCGTGT 3121 TCATCTTTCC ACCTAGCGAC GAGCAGCTGA AGTCTGGCAC AGCTAGCGTC GTGTGCCTGC 3181 TGAACAACTT CTACCCCAGA GAAGCCAAGG TGCAGTGGAA GGTGGACAAC GCCCTGCAGA 3241 GCGGCAATAG CCAAGAGAGC GTGACCGAGC AGGACAGCAA GGACTCCACC TATAGCCTGA 3301 GCAGCACACT GACCCTGAGC AAGGCCGACT ACGAGAAGCA CAAAGTGTAC GCCTGCGAAG 3361 TGACCCACCA GGGCCTTTCT AGCCCTGTGA CCAAGAGCTT CAACCGGGGC GAATGTTGAG 3421 aattcgatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga 3481 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt 3541 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt 3601 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg 3661 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg 3721 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc 3781 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat 3841 catcgtcctt tccttggctg ctcgcctatg ttgccacctg gattctgcgc gggacgtcct 3901 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg 3961 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg 4021 ccgcctcccc gcCTCGAGca tggcccaact tgtttattgc agcttataat ggttacaaat 4081 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg 4141 gtttgtccaa actcatcaat gtatcttatc atgtctggat ctccggacca cgtgcggacc 4201 gagcggccgc aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg 4261 ctcactgagg ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca 4321 gtgagcgagc gagcgcgcag ctgcctgcag g BRV-P-009 (SEQ ID NO: 170) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac tagtttaatt aaactagaca 181 gactgcagag ggccctgcgt atgagtgcaa gtgggtttta ggaccaggat gaggcggggt 241 gggggtgcct acctgacgac cgaccccgac ccactggaca agcacccaac ccccattccc 301 caaattgcgc atcccctatc agagaggggg aggggaaaca ggatgcggcg aggcgcgtcg 361 cgactgccag cttcagcacc gcggacagtg ccttcgcccc cgcctggcgg cgcgcgccac 421 cgccgcctca gcactgaagg cgcgctgacg tcactcgccg gtcccccgca aactcccctt481 cccggccacc ttggtcgcgt ccgcgccgcc gccggcccag ccggaccgca ccacgcgagg 541 cgcgagatag gggggcacgg gcgcgaccat ctgcgctgcg gcgccggcga ctcagcgctg 601 cctcagtctg cggtgggcag cggaggagtc gtgtcgtgcc tgagagcgca gtcgagagga 661 tccccgccac cATGGCGACG GGTTCAAGAA CTTCCCTACT TCTTGCATTT GGCCTGCTTT 721 GTTTGCCGTG GTTACAGGAG GGCTCGGCAC AGGTTCAGCT GCAACAGCCT GGCGCCGAAC 781 TTGTGAAACC TGGCGCCTCT GTGAAGATGA GCTGCAAGGC CAGCGGCTAC ACCTTCACCA 841 GCTACAACAT GCACTGGGTC AAGCAGACCC CTGGCAGAGG CCTGGAATGG ATCGGAGCCA 901 TCTATCCCGG CAACGGCGAC ACCTCCTACA ACCAGAAGTT CAAGGGCAAA GCCACACTGA 961 CCGCCGACAA GAGCAGCAGC ACAGCCTACA TGCAGCTGAG CAGCCTGACC AGCGAAGATA 1021 GCGCCGTGTA CTACTGCGCC AGAAGCACCT ATTACGGCGG CGACTGGTAC TTCAACGTGT 1081 GGGGAGCTGG CACCACCGTG ACAGTGTCTG CCGCCTCTAC AAAGGGCCCT AGCGTTTTCC 1141 CACTGGCTCC CAGCAGCAAG AGCACATCTG GTGGAACAGC CGCTCTGGGC TGCCTGGTCA 1201 AGGATTACTT TCCCGAGCCT GTGACCGTGT CCTGGAATAG CGGAGCACTG ACAAGCGGCG 1261 TGCACACCTT TCCAGCAGTG CTGCAAAGCA GCGGCCTGTA CTCTCTGAGC AGCGTGGTCA 1321 CAGTGCCTAG CTCTAGCCTG GGCACCCAGA CCTACATCTG CAATGTGAAC CACAAGCCTA 1381 GCAACACCAA GGTGGACAAG AAGGTGGAAC CCAAGAGCTG CGACAAGACC CACACCTGTC 1441 CTCCATGTCC TGCTCCAGAA CTGCTCGGCG GACCTTCCGT GTTCCTGTTT CCTCCAAAGC 1501 CTAAGGACAC CCTGATGATC AGCAGAACCC CTGAAGTGAC CTGCGTGGTG GTGGATGTGT 1561 CTCACGAGGA CCCCGAAGTG AAGTTCAATT GGTACGTGGA CGGCGTGGAA GTGCACAACG 1621 CCAAGACCAA GCCTAGAGAG GAACAGTACA ACAGCACCTA CAGAGTGGTG TCCGTGCTGA 1681 CCGTGCTGCA CCAGGATTGG CTGAACGGCA AAGAGTACAA GTGCAAGGTG TCCAACAAGG 1741 CCCTGCCTGC TCCTATCGAG AAAACCATCA GCAAGGCCAA GGGCCAGCCT AGGGAACCCC 1801 AGGTTTACAC ACTGCCTCCA AGCAGGGACG AGCTGACCAA GAATCAGGTG TCCCTGACCT 1861 GCCTCGTGAA GGGCTTCTAC CCTTCCGATA TCGCCGTGGA ATGGGAGAGC AATGGCCAGC 1921 CTGAGAACAA CTACAAGACA ACCCCTCCTG TGCTGGACAG CGACGGCTCA TTCTTCCTGT 1981 ACAGCAAGCT GACAGTGGAC AAGTCCAGAT GGCAGCAGGG CAACGTGTTC TCCTGCAGCG 2041 TGATGCACGA GGCCCTGCAC AACCACTACA CCCAGAAAAG CCTGTCTCTG AGCCCCGGCA 2101 AGCGAAAAAG AAGATCAGGT TCGGGTGCGC CAGTAAAGCA GACATTAAAC TTTGATTTGC 2161 TGAAACTTGC AGGTGATGTA GAGTCAAATC CAGGTCCAAT GGCAACAGGG AGCCGAACCT 2221 CTCTGCTCCT TGCTTTCGGG CTCCTTTGCC TACCGTGGCT CCAAGAGGGC TCGGCACAGA 2281 TCGTGCTGTC TCAGTCTCCT GCCATCCTGT CTGCTAGCCC TGGCGAGAAA GTGACCATGA 2341 CCTGTAGAGC CAGCAGCAGC GTGTCCTACA TCCACTGGTT CCAGCAGAAG CCTGGCAGCA 2401 GCCCTAAGCC TTGGATCTAC GCCACAAGCA ATCTGGCCAG CGGCGTGCCA GTCAGATTTT 2461 CTGGCTCTGG CAGCGGCACC AGCTACAGCC TGACAATCAG CAGAGTGGAA GCCGAGGATG 2521 CCGCCACCTA CTACTGTCAG CAGTGGACCA GCAATCCTCC TACCTTTGGC GGAGGCACCA 2581 AGCTGGAAAT CAAGCGGACA GTGGCCGCTC CTAGCGTGTT CATCTTTCCA CCTAGCGACG 2641 AGCAGCTGAA GTCTGGCACA GCTAGCGTCG TGTGCCTGCT GAACAACTTC TACCCCAGAG 2701 AAGCCAAGGT GCAGTGGAAG GTGGACAACG CCCTGCAGAG CGGCAATAGC CAAGAGAGCG 2761 TGACCGAGCA GGACAGCAAG GACTCCACCT ATAGCCTGAG CAGCACACTG ACCCTGAGCA 2821 AGGCCGACTA CGAGAAGCAC AAAGTGTACG CCTGCGAAGT GACCCACCAG GGCCTTTCTA 2881 GCCCTGTGAC CAAGAGCTTC AACCGGGGCG AGTGTTGAGa attcgatatc aagcttatcg 2941 ataatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg 3001 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc 3061 gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt 3121 tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca 3181 ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc 3241 ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc 3301 tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc 3361 tcgcctatgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc 3421 tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc 3481 ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg cCTCGAGcat 3541 ggcccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 3601 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 3661 tatcttatca tgtctggatc tccggaccac gtgcggaccg agcggccgca ggaaccccta 3721 gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca 3781 aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcagc 3841 tgcctgcaggBRV-P-010 (SEQ ID NO: 171) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgTaacat atcctggtgt ggagtagggg 181 acgctgctct gacagaggct cgggggcctg agctggctct gtgagctggg gaggaggcag 241 acagccaggc cttgtctgca agcagacctg gcagcattgg gctggccgcc ccccagggcc 301 tcctcttcat gcccagtgaa tgactcacct tggcacagac acaatgttcg gggtgggcac 361 agtgcctgct tcccgccgca ccccagcccc cctcaaatgc cttccgagaa gcccattgag 421 cagggggctt gcattgcacc ccagcctgac agcctggcat cttgggataa aagcagcaca 481 gccccctagg ggctgccctt gctgtgtggc gccaccggcg gtggagaaca aggctctatt 541 cagcctgtgc ccaggaaagg ggatcagggg atgcccaggc atggacagtg ggtggcaggg 601 ggggagagga gggctgtctg cttcccagaa gtccaaggac acaaatgggt gaggggagag 661 ctctccccat agctgggctg cggcccaacc ccaccccctc aggctatgcc agggggtgtt 721 gccaggggca cccgggcatc gccagtctag cccactcctt cataaagccc tcgTatccca 781 ggagcgagca gagccagagc aggttggaga ggagacgcat cacctctgct gcttgGTCGA 841 CCCTCAGCGC CACcATGGCG ACGGGTTCAA GAACTTCCCT ACTTCTTGCA TTTGGCCTGC 901 TTTGTTTGCC GTGGTTACAG GAGGGCTCGG CACAGGTTCA GCTGCAACAG CCTGGCGCCG 961 AACTTGTGAA ACCTGGCGCC TCTGTGAAGA TGAGCTGCAA GGCCAGCGGC TACACCTTCA 1021 CCAGCTACAA CATGCACTGG GTCAAGCAGA CCCCTGGCAG AGGCCTGGAA TGGATCGGAG 1081 CCATCTATCC CGGCAACGGC GACACCTCCT ACAACCAGAA GTTCAAGGGC AAAGCCACAC 1141 TGACCGCCGA CAAGAGCAGC AGCACAGCCT ACATGCAGCT GAGCAGCCTG ACCAGCGAAG 1201 ATAGCGCCGT GTACTACTGC GCCAGAAGCA CCTATTACGG CGGCGACTGG TACTTCAACG 1261 TGTGGGGAGC TGGCACCACC GTGACAGTGT CTGCCGCCTC TACAAAGGGC CCTAGCGTTT 1321 TCCCACTGGC TCCCAGCAGC AAGAGCACAT CTGGTGGAAC AGCCGCTCTG GGCTGCCTGG 1381 TCAAGGATTA CTTTCCCGAG CCTGTGACCG TGTCCTGGAA TAGCGGAGCA CTGACAAGCG 1441 GCGTGCACAC CTTTCCAGCA GTGCTGCAAA GCAGCGGCCT GTACTCTCTG AGCAGCGTGG 1501 TCACAGTGCC TAGCTCTAGC CTGGGCACCC AGACCTACAT CTGCAATGTG AACCACAAGC 1561 CTAGCAACAC CAAGGTGGAC AAGAAGGTGG AACCCAAGAG CTGCGACAAG ACCCACACCT 1621 GTCCTCCATG TCCTGCTCCA GAACTGCTCG GCGGACCTTC CGTGTTCCTG TTTCCTCCAA 1681 AGCCTAAGGA CACCCTGATG ATCAGCAGAA CCCCTGAAGT GACCTGCGTG GTGGTGGATG 1741 TGTCTCACGA GGACCCCGAA GTGAAGTTCA ATTGGTACGT GGACGGCGTG GAAGTGCACA 1801 ACGCCAAGAC CAAGCCTAGA GAGGAACAGT ACAACAGCAC CTACAGAGTG GTGTCCGTGC 1861 TGACCGTGCT GCACCAGGAT TGGCTGAACG GCAAAGAGTA CAAGTGCAAG GTGTCCAACA 1921 AGGCCCTGCC TGCTCCTATC GAGAAAACCA TCAGCAAGGC CAAGGGCCAG CCTAGGGAAC 1981 CCCAGGTTTA CACACTGCCT CCAAGCAGGG ACGAGCTGAC CAAGAATCAG GTGTCCCTGA 2041 CCTGCCTCGT GAAGGGCTTC TACCCTTCCG ATATCGCCGT GGAATGGGAG AGCAATGGCC 2101 AGCCTGAGAA CAACTACAAG ACAACCCCTC CTGTGCTGGA CAGCGACGGC TCATTCTTCC 2161 TGTACAGCAA GCTGACAGTG GACAAGTCCA GATGGCAGCA GGGCAACGTG TTCTCCTGCA 2221 GCGTGATGCA CGAGGCCCTG CACAACCACT ACACCCAGAA AAGCCTGTCT CTGAGCCCCG 2281 GCAAGCGAAA AAGAAGATCA GGTTCGGGTG CGCCAGTAAA GCAGACATTA AACTTTGATT 2341 TGCTGAAACT TGCAGGTGAT GTAGAGTCAA ATCCAGGTCC AATGGCAACA GGGAGCCGAA 2401 CCTCTCTGCT CCTTGCTTTC GGGCTCCTTT GCCTACCGTG GCTCCAAGAG GGCTCGGCAC 2461 AGATCGTGCT GTCTCAGTCT CCTGCCATCC TGTCTGCTAG CCCTGGCGAG AAAGTGACCA 2521 TGACCTGTAG AGCCAGCAGC AGCGTGTCCT ACATCCACTG GTTCCAGCAG AAGCCTGGCA 2581 GCAGCCCTAA GCCTTGGATC TACGCCACAA GCAATCTGGC CAGCGGCGTG CCAGTCAGAT 2641 TTTCTGGCTC TGGCAGCGGC ACCAGCTACA GCCTGACAAT CAGCAGAGTG GAAGCCGAGG 2701 ATGCCGCCAC CTACTACTGT CAGCAGTGGA CCAGCAATCC TCCTACCTTT GGCGGAGGCA 2761 CCAAGCTGGA AATCAAGCGG ACAGTGGCCG CTCCTAGCGT GTTCATCTTT CCACCTAGCG 2821 ACGAGCAGCT GAAGTCTGGC ACAGCTAGCG TCGTGTGCCT GCTGAACAAC TTCTACCCCA 2881 GAGAAGCCAA GGTGCAGTGG AAGGTGGACA ACGCCCTGCA GAGCGGCAAT AGCCAAGAGA 2941 GCGTGACCGA GCAGGACAGC AAGGACTCCA CCTATAGCCT GAGCAGCACA CTGACCCTGA 3001 GCAAGGCCGA CTACGAGAAG CACAAAGTGT ACGCCTGCGA AGTGACCCAC CAGGGCCTTT 3061 CTAGCCCTGT GACCAAGAGC TTCAACCGGG GCGAGTGTTG AGaattcgat atcaagctta 3121 tcgataatca acctctggat tacaaaattt gtgaaagatt gactggtatt cttaactatg 3181 ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat gctattgctt 3241 cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct ctttatgagg3301 agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct gacgcaaccc 3361 ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc gctttccccc 3421 tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg acaggggctc 3481 ggctgttggg cactgacaat tccgtggtgt tgtcggggaa atcatcgtcc tttccttggc 3541 tgctcgccta tgttgccacc tggattctgc gcgggacgtc cttctgctac gtcccttcgg 3601 ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg cctcttccgc 3661 gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc ccgcCTCGAG 3721 catggcccaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa 3781 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca 3841 atgtatctta tcatgtctgg atctccggac cacgtgcgga ccgagcggcc gcaggaaccc 3901 ctagtgatgg agttggccac tccctctctg cgcgctcgct cgctcactga ggccgggcga 3961 ccaaaggtcg cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc 4021 agctgcctgc agg BRV-P-011 (SEQ ID NO: 172) 1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc 61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca 121 actccatcac taggggttcc tgcggccgca cgcgtgttac taggttctag aaccggtgac 181 gtctcccatg gtgaagcttg gatctgaatt cggtacctag ttattaatag taatcaatta 241 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 301 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 361 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 421 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 481 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 541 cttggcagta catctacgta ttagtcatcg ctattaccat ggtcgaggtg agccccacgt 601 tctgcttcac tctccccatc tcccccccct ccccaccccc aattttgtat ttatttattt 661 tttaattatt ttgtgcagcg atgggggcgg gggggggggg ggggcgcgcg ccaggcgggg 721 cggggcgggg cgaggggcgg ggcggggcga ggcggagagg tgcggcggca gccaatcaga 781 gcggcgcgct ccgaaagttt ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa 841 agcgaagcgc gcggcgggcg ggagtcgctg cgacgctgcc ttcgccccgt gccccgctcc 901 gccgccgcct cgcgccgccc gccccggctc tgactgaccg cgttactccc acaggtgagc 961 gggcgggacg gcccttctcc tccgggctgt aattagcgct tggtttaatg acggcttgtt 1021 tcttttctgt ggctgcgtga aagccttgag gggctccggg agctagagcc tctgctaacc 1081 atgttcatgc cttcttcttt ttcctacagc tcctgggcaa cgtgctggtt attgtgctgt 1141 ctcatcattt tggcaaagaa ttcctcgaag atccgaaggg aaagtcttcc acgactgtgg 1201 gatccccgcc accATGGCGA CGGGTTCAAG AACTTCCCTA CTTCTTGCAT TTGGCCTGCT 1261 TTGTTTGCCG TGGTTACAGG AGGGCTCGGC ACAGGTTCAG CTGCAACAGC CTGGCGCCGA 1321 ACTTGTGAAA CCTGGCGCCT CTGTGAAGAT GAGCTGCAAG GCCAGCGGCT ACACCTTCAC 1381 CAGCTACAAC ATGCACTGGG TCAAGCAGAC CCCTGGCAGA GGCCTGGAAT GGATCGGAGC 1441 CATCTATCCC GGCAACGGCG ACACCTCCTA CAACCAGAAG TTCAAGGGCA AAGCCACACT 1501 GACCGCCGAC AAGAGCAGCA GCACAGCCTA CATGCAGCTG AGCAGCCTGA CCAGCGAAGA 1561 TAGCGCCGTG TACTACTGCG CCAGAAGCAC CTATTACGGC GGCGACTGGT ACTTCAACGT 1621 GTGGGGAGCT GGCACCACCG TGACAGTGTC TGCCGCCTCT ACAAAGGGCC CTAGCGTTTT 1681 CCCACTGGCT CCCAGCAGCA AGAGCACATC TGGTGGAACA GCCGCTCTGG GCTGCCTGGT 1741 CAAGGATTAC TTTCCCGAGC CTGTGACCGT GTCCTGGAAT AGCGGAGCAC TGACAAGCGG 1801 CGTGCACACC TTTCCAGCAG TGCTGCAAAG CAGCGGCCTG TACTCTCTGA GCAGCGTGGT 1861 CACAGTGCCT AGCTCTAGCC TGGGCACCCA GACCTACATC TGCAATGTGA ACCACAAGCC 1921 TAGCAACACC AAGGTGGACA AGAAGGTGGA ACCCAAGAGC TGCGACAAGA CCCACACCTG 1981 TCCTCCATGT CCTGCTCCAG AACTGCTCGG CGGACCTTCC GTGTTCCTGT TTCCTCCAAA 2041 GCCTAAGGAC ACCCTGATGA TCAGCAGAAC CCCTGAAGTG ACCTGCGTGG TGGTGGATGT 2101 GTCTCACGAG GACCCCGAAG TGAAGTTCAA TTGGTACGTG GACGGCGTGG AAGTGCACAA 2161 CGCCAAGACC AAGCCTAGAG AGGAACAGTA CAACAGCACC TACAGAGTGG TGTCCGTGCT 2221 GACCGTGCTG CACCAGGATT GGCTGAACGG CAAAGAGTAC AAGTGCAAGG TGTCCAACAA 2281 GGCCCTGCCT GCTCCTATCG AGAAAACCAT CAGCAAGGCC AAGGGCCAGC CTAGGGAACC 2341 CCAGGTTTAC ACACTGCCTC CAAGCAGGGA CGAGCTGACC AAGAATCAGG TGTCCCTGAC 2401 CTGCCTCGTG AAGGGCTTCT ACCCTTCCGA TATCGCCGTG GAATGGGAGA GCAATGGCCA 2461 GCCTGAGAAC AACTACAAGA CAACCCCTCC TGTGCTGGAC AGCGACGGCT CATTCTTCCT2521 GTACAGCAAG CTGACAGTGG ACAAGTCCAG ATGGCAGCAG GGCAACGTGT TCTCCTGCAG 2581 CGTGATGCAC GAGGCCCTGC ACAACCACTA CACCCAGAAA AGCCTGTCTC TGAGCCCCGG 2641 CAAGCGAAAA AGAAGATCAG GTTCGGGTGC GCCAGTAAAG CAGACATTAA ACTTTGATTT 2701 GCTGAAACTT GCAGGTGATG TAGAGTCAAA TCCAGGTCCA ATGGCAACAG GGAGCCGAAC 2761 CTCTCTGCTC CTTGCTTTCG GGCTCCTTTG CCTACCGTGG CTCCAAGAGG GCTCGGCACA 2821 GATCGTGCTG TCTCAGTCTC CTGCCATCCT GTCTGCTAGC CCTGGCGAGA AAGTGACCAT 2881 GACCTGTAGA GCCAGCAGCA GCGTGTCCTA CATCCACTGG TTCCAGCAGA AGCCTGGCAG 2941 CAGCCCTAAG CCTTGGATCT ACGCCACAAG CAATCTGGCC AGCGGCGTGC CAGTCAGATT 3001 TTCTGGCTCT GGCAGCGGCA CCAGCTACAG CCTGACAATC AGCAGAGTGG AAGCCGAGGA 3061 TGCCGCCACC TACTACTGTC AGCAGTGGAC CAGCAATCCT CCTACCTTTG GCGGAGGCAC 3121 CAAGCTGGAA ATCAAGCGGA CAGTGGCCGC TCCTAGCGTG TTCATCTTTC CACCTAGCGA 3181 CGAGCAGCTG AAGTCTGGCA CAGCTAGCGT CGTGTGCCTG CTGAACAACT TCTACCCCAG 3241 AGAAGCCAAG GTGCAGTGGA AGGTGGACAA CGCCCTGCAG AGCGGCAATA GCCAAGAGAG 3301 CGTGACCGAG CAGGACAGCA AGGACTCCAC CTATAGCCTG AGCAGCACAC TGACCCTGAG 3361 CAAGGCCGAC TACGAGAAGC ACAAAGTGTA CGCCTGCGAA GTGACCCACC AGGGCCTTTC 3421 TAGCCCTGTG ACCAAGAGCT TCAACCGGGG CGAGTGTTGA Gaattcgata tcaagcttat 3481 cgataatcaa cctctggatt acaaaatttg tgaaagattg actggtattc ttaactatgt 3541 tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg ctattgcttc 3601 ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc tttatgagga 3661 gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc 3721 cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg ctttccccct 3781 ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga caggggctcg 3841 gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct ttccttggct 3901 gctcgcctat gttgccacct ggattctgcg cgggacgtcc ttctgctacg tcccttcggc 3961 cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg 4021 tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc cgcCTCGAGc 4081 atggcccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa 4141 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa 4201 tgtatcttat catgtctgga tctccggacc acgtgcggac cgagcggccg caggaacccc 4261 tagtgatgga gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac 4321 caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca 4381 gctgcctgca gg

Claims

CLAIMS What is claimed is:

1. An adeno-associated viral (AAV) vector comprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain.

2. The AAV vector of claim 1, wherein the AAV vector comprises one or more modifications to the capsid which increase the efficiency with which the AAV vector crosses the blood brain barrier in a subject relative to an AAV vector comprising an unmodified capsid.

3. The AAV vector of claim 2, wherein the one or more modifications comprises a targeting peptide in the capsid.

4. The AAV vector of claim 3, wherein the targeting peptide comprises TVSALFK (SEQ ID NO: 96) or TVSALK (SEQ ID NO: 92) or a sequence having 1, 2 or 3 substitutions, insertions or deletions relative thereto.

5. The AAV vector of any one of claims 1-4, wherein the capsid comprises an AAV9 capsid or an rh10 capsid.

6. The AAV vector of claim 5, wherein the targeting peptide is inserted between amino acids 588 and 589 relative to SEQ ID NO:

1.

7. The AAV vector of any one of claims 1-6, wherein the antigen binding domain comprises an antibody, an scFv, an Fv, a Fab fragment, a Fab’ fragment, a F(abƍ)2fragment, a diabody, a single-chain antibody, or a single domain antibody.

8. The AAV vector of any one of claims 1-7, wherein the CD20 antigen binding domain is isolated or derived from rituximab, ofatumumab, obinutuzumab, ibritumomab, ocrelizumab or ublituximab.

9. The AAV vector of any one of claims 1-7, wherein the CD20 antigen binding domain comprises a heavy chain variable region and a light chain variable region.

10. The AAV vector of claim 9, wherein the heavy chain variable region comprises a complementarity determining region 1 (CDR-H1) comprising a sequence of SYNMH (SEQ ID NO: 2), a CDR-H2 comprising a sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 3), and a CDR-H3 comprising a sequence of STYYGGDWYFNV (SEQ ID NO: 4), orsequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a CDR-L1 comprising a sequence of RASSSVSYIH (SEQ ID NO: 5), a CDR-L2 comprising a sequence of ATSNLAS (SEQ ID NO: 6) and a CDR-L3 comprising a sequence of QQWTSNPPT (SEQ ID NO: 7), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

11. The AAV vector of claim 10, wherein the heavy chain variable region comprises a sequence of QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGN GDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNV WGAGTTVTVS (SEQ ID NO: 20), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

12. The AAV vector of claim 10 or 11, wherein the light chain variable region comprises a sequence of QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVA (SEQ ID NO: 21), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

13. The AAV vector of any one of claims 10-12, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 28, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 29, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

14. The AAV vector of claim 9, wherein the heavy chain variable region comprises a CDR-H1 comprising a sequence of DYAMH (SEQ ID NO: 8), a CDR-H2 comprising a sequence of TISWNSGSIGYADSVKG (SEQ ID NO: 9), and a CDR-H3 comprising a sequence ofDIQYGNYYYGMDV (SEQ ID NO: 10), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a CDR-L1 comprising a sequence of RASQSVSSYLA (SEQ ID NO: 11), a CDR-L2 comprising a sequence of DASNRAT (SEQ ID NO: 12) and a CDR-L3 comprising a sequence of QQRSNWPIT (SEQ ID NO: 13), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

15. The AAV vector of claim 14, wherein the heavy chain variable region comprises a sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNS GSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMD VWGQGTTVTVS (SEQ ID NO: 22), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

16. The AAV vector of claim 14 or 15, wherein the light chain variable region comprises a sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVA (SEQ ID NO: 23), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

17. The AAV vector of any one of claims 14-16, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 30, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 31, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

18. The AAV vector of claim 9, wherein the heavy chain variable region comprises a CDR-H1 comprising a sequence of YSWIN (SEQ ID NO: 14), a CDR-H2 comprising a sequence of RIFPGDGDTDYNGKFKG (SEQ ID NO: 15), and a CDR-H3 comprising a sequence ofNVFDGYWLVY (SEQ ID NO: 16), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a CDR-L1 comprising a sequence of RSSKSLLHSNGITYLY (SEQ ID NO: 17), a CDR-L2 comprising a sequence of QMSNLVS (SEQ ID NO: 18) and a CDR-L3 comprising a sequence of AQNLELPYT (SEQ ID NO: 19), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

19. The AAV vector of claim 18, wherein the heavy chain variable region comprises a sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGD GDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG QGTLVTVS (SEQ ID NO: 24),or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

20. The AAV vector of claim 18 or 19, wherein the light chain variable region comprises a sequence of DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNL VSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVA (SEQ ID NO: 25), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

21. The AAV vector of any one of claims 18-20, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 32, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 33, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

22. The AAV vector of any one of claims 1-21, wherein the CD20 antigen binding domain comprises a heavy chain and a light chain, and wherein the heavy chain and light chain are separated by a linker comprising one or more protease cleavage sites, one or more self- cleaving peptides, and / or an internal ribosome entry site (IRES).

23. The AAV vector of claim 22, wherein the one or more protease cleavage sites comprise a furin cleavage site.

24. The AAV of claim 22 or 23, wherein the self-cleaving peptide comprises an E2A peptide, a P2A peptide, a T2A peptide or an F2A peptide.

25. The AAV vector of any one of claims 22-24, wherein the linker comprises a sequence of RKRRSGSGAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 64).

26. The AAV vector of any one of claims 1-25, wherein the sequence encoding the CD20 antigen binding domain is operably linked to a promoter capable of expressing the CD20 antigen binding domain in a central nervous system cell, an immune cell, or a cell derived from a central nervous system cell or immune cell.

27. The AAV vector of claim 26, wherein the central nervous system cell comprises a brain cell and / or spinal cord cell.

28. The AAV vector of claim 26 or 27, wherein the central nervous system cell comprises an astrocyte, oligodendrocyte or a neuron.

29. The AAV vector of claim 26, wherein the cell derived from an immune cell is a primary central nervous system lymphoma (PCNSL) cell.

30. The AAV vector of any one of claims 26-29, wherein the promoter is selected from the group consisting of a CAG promoter, an hSyn promoter, a GFAP promoter, and a CBA promoter.

31. The AAV vector of claim 30, wherein: a. the CAG promoter comprises a sequence of SEQ ID NO: 40, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; b. the hSyn promoter comprises a sequence of SEQ ID NO: 41, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; c. the GFAP promoter comprises a sequence of SEQ ID NO: 42, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; or d. the CBA promoter comprises a sequence of SEQ ID NO: 43, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

32. The AAV vector of any one of claims 1-31, comprising a sequence encoding at least one signal peptide operably linked to the sequence encoding the CD20 antigen binding domain.

33. The AAV vector of claim 32, wherein the CD20 antigen comprises a heavy chain and a light chain, and the sequence encoding the heavy chain and light chain are both operably linked to sequences encoding signal peptides.

34. The AAV vector of claim 33, wherein the sequences encoding the signal peptides are 5’ of the sequences encoding the heavy and light chains.

35. The AAV vector of any one of claims 32-34, wherein the at least one signal peptide is isolated or derived from HGH, IgKVIII, H7, GRN, IL2 or L2.

36. The AAV vector of claim 35, wherein: a. the signal peptide isolated or derived from HGH comprises a sequence of MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 44), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; b. the signal peptide isolated or derived from IgKVIII comprises a sequence of MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 48), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; c. the signal peptide isolated or derived from H7 comprises a sequence of MEFGLSWVFLVALFRGVQC (SEQ ID NO: 45), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; d. the signal peptide isolated or derived from GRN comprises a sequence of MWTLVSWVALTAGLVAG (SEQ ID NO: 49), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; e. the signal peptide isolated or derived from IL2 comprises a sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 47), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; orf. the signal peptide isolated or derived from L2 comprises a sequence of MKYLLPTAAAGLLLLAAQPAMA (SEQ ID NO: 46), or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

37. The AAV vector of any one of claims 1-36, wherein the polynucleotide comprises one or more microRNA (miRNA) targeting sequences that reduce expression of the CD20 antigen binding domain in the liver and / or antigen present cells (APC) of the subject when compared to an AAV vector that does not include the one or more miRNA targeting sequences.

38. The AAV vector of claim 37, wherein the one or more microRNA targeting sequences comprise a miR122 sequence, a miR-142-3p sequence, or a combination thereof.

39. The AAV vector of claim 37 or 38, wherein the miRNA targeting site comprises a sequence selected from the group consisting of SEQ ID NOS: 59-61.

40. The AAV vector of any one of claims 37-39, wherein the one or more miRNA targeting sequences are 3’ of the sequence encoding the CD20 antigen binding domain.

41. The AAV vector of any one of claims 1-40, wherein the polynucleotide comprises a Woodchuck Hepatitis Posttranscriptional Regulatory Element (WPRE).

42. The AAV vector of claim 41, wherein the WPRE comprises a sequence of SEQ ID NO: 62, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

43. The AAV vector of any one of claims 1-42, comprising a polyadenylation signal 3’ of the sequence encoding the CD20 antigen binding domain.

44. The AAV vector of claim 43, wherein the polyadenylation signal comprises a sequence of SEQ ID NO: 63, or a sequence having at least 80%, %, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

45. The AAV vector of any one of claims 1-44, comprising 5’ inverted terminal repeat and a 3’ inverted terminal repeat isolated or derived from AAV2.

46. The AAV vector of any one of claims 1-45, wherein the polynucleotide comprises, from 5’ to 3’: a. an 5’ AAV2 inverted terminal repeat (ITR); b. a promoter; c. a sequence encoding a first signal peptide;d. a sequence encoding a CD20 antigen binding domain heavy chain; e. a sequence encoding a linker comprising one or more protease cleavage sites and / or self-cleaving peptides, or an internal ribosome entry site (IRES); f. a sequence encoding a second signal peptide; g. a sequence encoding a CD20 antigen binding domain light chain; and h. a 3’ AAV2 ITR.

47. The AAV vector of any one of claims 1-46, wherein the polynucleotide comprises, from 5’ to 3’: a. an 5’ AAV2 inverted terminal repeat (ITR); b. a promoter; c. a sequence encoding a first signal peptide; d. a sequence encoding a CD20 antigen binding domain light chain; e. a linker comprising one or more protease cleavage sites and / or self-cleaving peptides, or an internal ribosome entry site (IRES); f. a sequence encoding a second signal peptide; g. a sequence encoding a CD20 antigen binding domain heavy chain; and h. a 3’ AAV2 ITR.

48. The AAV vector of claim 46 or 47, comprising, between the sequence encoding the CD20 antigen binding domain light chain or heavy chain and the 3’ AAV2 ITR, one or more microRNA targeting sequences, a WPRE and / or a polyadenylation signal.

49. A pharmaceutical composition comprising the AAV of any one of claims 1-48 and a pharmaceutically acceptable carrier, diluent or excipient.

50. A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of the AAV vector of any one of claims 1-48 or the pharmaceutical composition of claim 49.

51. The AV vector of any one of claims 1-48 or the pharmaceutical composition of claim 49, for use in treating a cancer in a subject in need thereof.

52. The AV vector of any one of claims 1-48, for use in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof.

53. A method of treating a cancer in a subject, comprising administering a therapeutically effective amount of a composition comprising an adeno-associated viral (AAV) vectorcomprising a capsid and a polynucleotide comprising a sequence encoding a CD20 antigen binding domain.

54. The method of claim 53, wherein the AAV vector comprises one or more modifications to the capsid which increase the efficiency with which the AAV vector crosses the blood brain barrier relative to an AAV vector comprising an unmodified capsid.

55. The method of claim 54, wherein the one or more modifications comprises a targeting peptide in the capsid.

56. The method of claim 55, wherein the targeting peptide comprises TVSALFK (SEQ ID NO: 96) or TVSALK (SEQ ID NO: 92) or a sequence having 1, 2 or 3 substitutions, insertions or deletions relative thereto.

57. The method of any one of claims 52-55, wherein the capsid comprises an AAV9 capsid or an rh10 capsid.

58. The method claim 56, wherein the targeting peptide is inserted between amino acids 588 and 589 relative to SEQ ID NO:

1.

59. The method any one of claims 53-58, wherein the antigen binding domain comprises an antibody, an scFv, an Fv, a Fab fragment, a Fab’ fragment, a F(abƍ)2fragment, a diabody, a single-chain antibody, or a domain antibody.

60. The method of any one of claims 53-59, wherein the CD20 antigen binding domain is isolated or derived from rituximab, ofatumumab, obinutuzumab, ibritumomab, ocrelizumab or ublituximab.

61. The method of any one of claims 53-59, wherein the CD20 antigen binding domain comprises a heavy chain variable region and a light chain variable region.

62. The method of claim 61, wherein the heavy chain variable region comprises a complementarity determining region 1 (CDR-H1) comprising a sequence of SYNMH (SEQ ID NO: 2), a CDR-H2 comprising a sequence of AIYPGNGDTSYNQKFKG (SEQ ID NO: 3), and a CDR-H3 comprising a sequence of STYYGGDWYFNV (SEQ ID NO: 4), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a CDR-L1 comprising a sequence of RASSSVSYIH (SEQ ID NO: 5), a CDR-L2 comprising a sequence of ATSNLAS (SEQ ID NO: 6) and a CDR-L3 comprising a sequence of QQWTSNPPT (SEQ ID NO: 7), orsequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

63. The method of claim 62, wherein the heavy chain variable region comprises a sequence of QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGN GDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNV WGAGTTVTVS (SEQ ID NO: 20), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

64. The method of claim 62 or 63, wherein the light chain variable region comprises a sequence of QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVP VRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVA (SEQ ID NO: 21), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

65. The method of any one of claims 62-64, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 28, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 29, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

66. The method of claim 61, wherein the heavy chain variable region comprises a CDR-H1 comprising a sequence of DYAMH (SEQ ID NO: 8), a CDR-H2 comprising a sequence of TISWNSGSIGYADSVKG (SEQ ID NO: 9), and a CDR-H3 comprising a sequence of DIQYGNYYYGMDV (SEQ ID NO: 10), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a CDR-L1 comprising a sequence of RASQSVSSYLA (SEQ ID NO: 11), a CDR-L2 comprising a sequence of DASNRAT (SEQ ID NO: 12) and a CDR-L3 comprising a sequence of QQRSNWPIT (SEQ ID NO: 13), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

67. The method of claim 66, wherein the heavy chain variable region comprises a sequence of EVQLVESGGGLVQPGRSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNS GSIGYADSVKGRFTISRDNAKKSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMD VWGQGTTVTVS (SEQ ID NO: 22), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

68. The method of claim 66 or 67, wherein the light chain variable region comprises a sequence of EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIKRTVA (SEQ ID NO: 23), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

69. The method of any one of claims 66-68, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 30, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 31, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

70. The method of claim 61, wherein the heavy chain variable region comprises a CDR-H1 comprising a sequence of YSWIN (SEQ ID NO: 14), a CDR-H2 comprising a sequence of RIFPGDGDTDYNGKFKG (SEQ ID NO: 15), and a CDR-H3 comprising a sequence of NVFDGYWLVY (SEQ ID NO: 16), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences; and wherein the light chain variable region comprises a sequence of CDR-L1 of RSSKSLLHSNGITYLY (SEQ ID NO: 17), a CDR-L2 comprising a sequence of QMSNLVS (SEQ ID NO: 18) and a CDR-L3 comprising a sequence of AQNLELPYT (SEQ ID NO: 19), or sequences having 1, 2 or 3 substitutions, insertions or deletions relative to any of the foregoing sequences.

71. The method of claim 70, wherein the heavy chain variable region comprises a sequence of QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWG QGTLVTVS (SEQ ID NO: 24),or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

72. The method of claim 70 or 71, wherein the light chain variable region comprises a sequence of DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNL VSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTVA (SEQ ID NO: 25), or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

73. The method of any one of claims 70-72, wherein the CD20 antigen binding domain comprises: a. a heavy chain comprising a sequence of SEQ ID NO: 32, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto; and b. a light chain comprising a sequence of SEQ ID NO: 33, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

74. The method of any one of claims 53-73, wherein the CD20 antigen binding domain comprises a heavy chain and a light chain, and wherein the heavy chain and light chain are separated by a linker comprising one or more protease cleavage sites, one or more self- cleaving peptides, and / or an internal ribosome entry site (IRES).

75. The method of claim 74, wherein the one or more protease cleavage sites comprise a furin cleavage site.

76. The method of claim 74 or 75, wherein the self-cleaving peptide comprises an E2A peptide, a P2A peptide, a T2A peptide or an F2A peptide.

77. The method of any one of claims 74-76, wherein the linker comprises a sequence of RKRRSGSGAPVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 64).

78. The method of any one of claims 53-77, wherein the sequence encoding the CD20 antigen binding domain is operably linked to a promoter capable of expressing the CD20 antigen binding domain in a central nervous system cell, an immune cell, or a cell derived from a central nervous system cell or immune cell.

79. The method of claim 78, wherein the central nervous system cell is a brain cell and / or spinal cord cell.

80. The method of claim 78 or 79, wherein the central nervous system cell comprises an astrocyte, oligodendrocyte or a neuron.

81. The method of claim 79, wherein the cell derived from an immune cell is a primary central nervous system lymphoma (PCNSL) cell or a secondary central nervous system lymphoma cell.

82. The method of any one of claims 78-81, wherein the promoter is selected from the group consisting of a CAG promoter, an hSyn promoter, a GFAP promoter, and a CBA promoter.

83. The method of any one of claims 53-82, wherein the AAV vector comprises a sequence encoding at least one signal peptide operably linked to the sequence encoding the CD20 antigen binding domain.

84. The method of claim 83, wherein the CD20 antigen comprises a heavy chain and a light chain, and the sequence encoding the heavy chain and light chain are both operably linked to sequences encoding signal peptides.

85. The method of claim 84, wherein the sequences encoding the signal peptides are 5’ of the sequences encoding the heavy and light chains.

86. The method of any one of claims 83-85, wherein the at least one signal peptide is isolated or derived from HGH, IgKVIII, H7, GRN, IL2 or L2.

87. The method of any one of claims 53-86, wherein the polynucleotide comprises one or more microRNA (miRNA) targeting sequences that reduce expression of the CD20 antigen binding domain in the liver and / or antigen present cells (APC) of the subject when compared to an AAV vector that does not include the one or more miRNA targeting sequences.

88. The method of claim 87, wherein the one or more microRNA targeting sequences comprise a miR122 sequence, a miR-142-3p sequence, or a combination thereof.

89. The method of claim 87 or 88, wherein the miRNA targeting site comprises a sequence selected from the group consisting of SEQ ID NOS: 59-61.

90. The method of any one of claims 87-89, wherein the one or more miRNA targeting sequences are 3’ of the sequence encoding the CD20 antigen binding domain.

91. The method of any one of claims 53-90, wherein the polynucleotide comprises a Woodchuck Hepatitis Posttranscriptional Regulatory Element (WPRE).

92. The method of any one of claims 53-91, comprising a polyadenylation signal 3’ of the sequence encoding the CD20 antigen binding domain.

93. The method of any one of claims 53-92, comprising 5’ inverted terminal repeat and a 3’ inverted terminal repeat isolated or derived from AAV2.

94. The method of any one of claims 53-93, wherein the cancer is a cancer of the central nervous system.

95. The method of claim 94, wherein the cancer of the central nervous system comprises primary central nervous system lymphoma (PCNSL) or secondary central nervous system lymphoma.

96. The method of any one of claims 53-95, wherein the composition comprising the AAV vector is administered via systemic or intrathecal administration.

97. The method of claim 96, wherein the intrathecal administration comprises administration to the cerebrospinal fluid (CSF).

98. The method of claim 96 or 97, wherein the intrathecal administration comprises intracerebroventricular (IVC) injection, intracisterna magna (ICM) injection or injection into the subarachnoid space of the spinal cord (IT) .

99. The method of any one of claims 53-98, wherein administration of the composition comprising the AAV vector: a. reduces a sign or a symptom of the cancer; b. slows or halts the progression of the cancer; c. reduces size and / or number of tumors of the cancer; and / or d. increases survival of the subject.

100. A kit comprising the AAV vector of any one of claims 1-48 or the pharmaceutical composition of claim 49, and instructions for use.

101. A polynucleotide, comprising: a. a 5’ inverted terminal repeat (ITR); b. a sequence encoding a promoter; c. a sequence encoding a CD20 antigen binding domain; d. a polyA signal; and e. a 3’ ITR.

102. The polynucleotide of claim 101, wherein the polynucleotide comprises a one or microRNA targeting sequences and / or a WPRE between the sequence encoding the CD20 antigen binding domain and the polyA signal.

103. The polynucleotide of claim 101 or 102, wherein the CD20 antigen binding domain comprises a heavy chain and a light chain separated by a linker comprising one or more protease cleavage sites, one or more self-cleaving peptides, and / or an IRES.

104. The polynucleotide of claim 103, wherein the CD20 antigen binding domain is encoded by a sequence comprising, from 5’ to 3’: i. a sequence encoding a first signal peptide; ii. a sequence encoding a heavy chain; iii. a sequence encoding the linker; iv. a sequence encoding a second signal peptide; and v. a sequence encoding a light chain.

105. The polynucleotide of claim 101, comprising a sequence selected from the group consisting of SEQ ID NOS: 162-172, or a sequence having at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98% or at least 99% identity thereto.

106. The polynucleotide of claim 101, comprising a sequence selected from the group consisting of SEQ ID NOS: 162-172.

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