US20160369298A1 - Synthetic combinatorial aav capsid library for targeted gene therapy - Google Patents

Synthetic combinatorial aav capsid library for targeted gene therapy Download PDF

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US20160369298A1
US20160369298A1 US15/024,431 US201415024431A US2016369298A1 US 20160369298 A1 US20160369298 A1 US 20160369298A1 US 201415024431 A US201415024431 A US 201415024431A US 2016369298 A1 US2016369298 A1 US 2016369298A1
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aav
sequence
vectors
capsid
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Damien Marsic
Sergei Zolotukhin
Mavis Agbandje-McKenna
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University of Florida Research Foundation Inc
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    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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Definitions

  • the present invention relates to the fields of molecular biology, medicine, and virology. More particularly, the invention relates to compositions and methods for producing modified adeno-associated virus (AAV) cap genes and combinatorial libraries of chimeric AAV vectors and virions; and in particular embodiments, for selecting for virions displaying cell-specific tropisms; and also for producing helper vectors that contain one or more modified, AAV cap genes.
  • AAV adeno-associated virus
  • the synthetic combinatorial AAV capsid libraries of the invention are useful in introducing into selected target host cells one or more nucleic acid molecules.
  • the viral vectors and genetic constructs disclosed herein are also useful in a variety of diagnostic and/or therapeutic gene-therapy regimens.
  • the invention provides novel AAV variants that transduce mouse liver nearly 200 times more efficiently than the corresponding, wild-type, unmodified AAV.
  • Adeno-associated virus is a single-stranded DNA virus belonging to the Parvoviridae family (Muzyczka and Berns, 2001).
  • AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, low immunogenicity, episomal localization and stable transgene expression.
  • significant limitations to the clinical use of AAV are its promiscuity and its susceptibility to neutralization by human antibodies (Jeune et al., 2013). Both of these limitations are determined by nature of the amino acid residues exposed at the surface of the capsid. Therefore, major efforts aiming at developing useful and effective gene therapy vectors have been devoted to obtaining and studying capsid variants (Wu et al., 2006).
  • the first approach was to study naturally occurring AAV isolates. So far, 13 serotypes have been formally characterized and hundreds of variant isolates have been sequenced. Additional capsid variation has been investigated through the generation of mosaics (viral particles made of capsid proteins from more than one serotype) (Hauck et al., 2003; Stachler and Bartlett, 2006; Gigout et al., 2005), chimeras (capsid proteins with domains from various origins) (Shen et al., 2007), and various substitutional or insertional mutants (Wu et al., 2000). However, the most significant advances are expected to result from directed evolution approaches through the development of capsid libraries.
  • Random display peptide libraries (Govindasamy et al., 2006) are limited to an insertion at one particular capsid location.
  • Libraries generated using error-prone PCR contain a very small fraction of gene variants encoding proteins that can fold properly and assemble into a functional capsid, due to the randomness of the mutations.
  • DNA shuffling and staggered extension processes are more efficient because they recombine naturally-occurring parental sequences and therefore are more likely to generate actual capsid variants. However, they can only recombine blocks of DNA as opposed to single nucleotide positions, which results in sequence bias (parental polymorphisms will tend to cluster together instead of being randomly distributed).
  • the present invention overcomes these and other limitations inherent in the art by providing novel capsid libraries, and methods for developing AAV variants that are capable of effectively targeting specific tissues and/or organs.
  • Various strategies have been described to generate AAV capsid libraries, including random peptide display, error-prone PCR or DNA shuffling.
  • the novel, gene synthesis-based approach described in the present application permits the alteration of only the amino acid residues that have side chains exposed to the capsid surface, as well as restricting diversity to naturally-occurring variants, thus improving compatibility with capsid structure and function and increasing the probability of generating useful variants.
  • a comparison of naturally-occurring AAV capsid protein sequences shows nine variable regions (commonly designated loops 1 to 9), while the rest of the capsid is highly conserved.
  • the DNA sequence encoding one of these regions also encodes another protein (Assembly-Activating Protein or AAP) in a different frame, which makes it unpractical to modify, AAP being required for proper assembly of functional viral particles. Therefore, the present capsid library construction involved the introduction of variations in the other eight loops only.
  • the invention provides a non-naturally occurring nucleic acid comprising: (a) a first nucleotide sequence encoding at least one AAV Rep protein; and (b) a second nucleotide sequence encoding at least one AAV Cap protein, wherein the second nucleotide sequence comprises: (i) a polynucleotide encoding a portion of a Cap protein found in an AAV of a first serotype but not in an AAV of a second serotype differing from the first serotype; and (ii) a polynucleotide encoding a portion of a Cap protein found in the AAV of the second serotype, but not in the AAV of the first serotype, wherein the non-naturally occurring nucleic acid further comprises a first AAV terminal repeat and a second AAV terminal repeat, and the first and second nucleotide sequences are interposed between the first and the second AAV terminal repeats.
  • the nucleic acid is comprised within a vector, and the AAV Rep protein is preferably from serotype 2.
  • the nucleic acid further may further include a third nucleotide sequence that encodes at least one molecule providing one or more helper function(s) preferably from an adenovirus, a herpesvirus, or a combination thereof.
  • the invention also provides a vector library that includes at least a first vector and a second vector, the first vector comprising a nucleic acid comprising: (a) a first nucleotide sequence encoding at least one AAV Rep protein; and (b) a second nucleotide sequence encoding at least one AAV Cap protein, wherein the second nucleotide sequence comprises: (i) a polynucleotide encoding a portion of a Cap protein found in an AAV of a first serotype but not in an AAV of a second serotype differing from the first serotype, and ((ii) a polynucleotide encoding a portion of a Cap protein found in the AAV of the second serotype, but not in the AAV of the first serotype, wherein the nucleic acid further comprises a first AAV TR and a second AAV TR, and the first and second nucleotide sequences are interposed between the first and the second AAV TRs, and the
  • the vector libraries of the present invention may be incorporated into at least one host cell, such as a mammalian or insect host cell.
  • the invention further provides an AAV virion that includes a nucleic acid comprising: (a) a first nucleotide sequence encoding at least one AAV Rep protein; (b) a second nucleotide sequence encoding at least one AAV Cap protein, wherein the second nucleotide sequence comprises (i) a polynucleotide encoding a portion of a Cap protein found in an AAV of a first serotype but not in an AAV of a second serotype differing from the first serotype, and (ii) a polynucleotide encoding a portion of a Cap protein found in the AAV of the second serotype, but not in the AAV of the first serotype; and (c) a first and a second AAV TR, wherein the first and the second nucleotide sequences are interposed between the first and the second AAV TRs.
  • a nucleic acid comprising: (a) a first nucleotide sequence encoding at
  • the AAV virion will further preferably include at least one AAV Cap protein encoded by the second nucleotide sequence, and in certain applications, the Cap protein is preferably a wild-type AAV Cap protein.
  • the second nucleotide sequence may further optionally include a polynucleotide that encodes a portion of a Cap protein found in an AAV of a fourth serotype but not found in a Cap protein of an AAV of the first, second or third serotypes, and may further still include a polynucleotide that encodes a portion of a Cap protein found in an AAV of a fifth serotype but not found in a Cap protein of an AAV of the first, second, third or fourth serotypes.
  • the AAV virions of the invention will include a second nucleotide sequence that further includes a polynucleotide that encodes a portion of a Cap protein found in an AAV of a sixth serotype but not found in a Cap protein of an AAV of the first, second, third, fourth or fifth serotypes.
  • the AAV virions of the invention may include a second nucleotide sequence that further includes a polynucleotide that encodes a portion of a Cap protein found in an AAV of a seventh serotype but not found in a Cap protein of an AAV of the first, second, third, fourth, fifth or sixth serotypes., or similarly, include a polynucleotide that encodes a portion of a Cap protein found in an AAV of an eight serotype but not found in a Cap protein of an AAV of the first, second, third, fourth, fifth, sixth or seventh serotypes.
  • the nucleic acid contained within the AAV vector will preferably further include a DNA sequence that encodes one or more diagnostic, therapeutic, and/or prophylactic molecules.
  • the nucleic acid preferably further includes one or more expression control sequence(s) that effect cell- or tissue-specific expression of the therapeutic molecule.
  • the expression control sequence includes one or more promoters operably linked to the sequence encoding the therapeutic molecule, which is preferably a polypeptide, a peptide, or an RNA.
  • FIG. 1A and FIG. 1B show the nucleotide sequence of CapLib variable regions (VRs) and corresponding amino acid diversity.
  • Each VR region shows a nucleotide sequence (lowercase letters) as designed and encoded by synthetic oligonucleotides, including degenerate positions (IUPAC nucleotide code) highlighted in yellow.
  • the corresponding amino acid sequence (UPPERCASE letters above nucleotide sequence) encoded by all possible codon combinations is shown in the line/s above. Amino acids highlighted in blue were selected for the inclusion into the library sequence because they were found in one or more naturally-occurring 150 variants used for the library design, while those that are not highlighted represent an additional amino acid diversity encoded by the degenerate nucleotides.
  • amino acid residues not encoded by the WT AAV2 sequence that were introduced during design to increase transduction efficiency (Y/F) or eliminate heparin binding (R/A).
  • the amino acid residues in logo style shows the alignment of the sequences deduced from the NextGen sequencing of the library viral DNA.
  • the X-axis designates residue position (VP1 numbering), and the Y-axis—the relative frequency of each amino acid at that position.
  • Amino acids are colored according to their chemical properties: polar amino acids are green; basic, blue; acidic, red; and hydrophobic amino acids are black;
  • FIG. 2 shows a schematic representation of the CapLib design and construction steps.
  • AAV2 capsid is shown at the top of the chart whereby numbers represent the respective amino acid residues.
  • Variable region (VR) sub-libraries were packaged and purified (indicated by differentially colored VRs). DNAs from these individual sub-libraries were purified and used construct a final combined library;
  • FIG. 3A , FIG. 3B , and FIG. 3C show variable regions (VRs) on the AAV2 capsid surface.
  • FIG. 3A AAV2 capsid full capsid (assembled from 60 VP3 monomers shown to the right), displaying the location of the eight VRs, colored as described below the image of the monomer .
  • FIG. 3B and FIG. 3C respectively: LiA and LiC full capsids variants and their respective monomers. The mutated residues and their positions are shown by color and by number, respectively;
  • FIG. 4A , FIG. 4B , FIG. 4C , FIG. 4D , and FIG. 4E show the analysis of packaged viral library complexity.
  • FIG. 4A Shannon entropy of the viral library computed from 840 individual protein sequences. The X-axis designates residue position (VP1 numbering) and the Y-axis—computed value of the entropy;
  • FIG. 4B Distribution of the distinct capsid sequences and their copy numbers;
  • FIG. 4C Distribution of the number of mutations in the sample of analyzed protein sequences;
  • FIG. 4D Distribution of the number of mutant VRs. The different combinations of mutated VRs are designated by the respective colors shown to the right of the graph.
  • FIG. 4E Comparison of expected and observed percentages of mutant residues.
  • the X-axis designates residue position (VP1 numbering); the respective VR boundaries are shown below amino acid numbers for better orientation.
  • the Y-axis designates the percentage of the expected (introduced by design) mutation (blue) vs experimentally deduced from the sequencing of the plasmid library (red) vs viral library (green).
  • the similitude of the graphs height at each position indicates a relative neutrality of this position for the capsid assembly/structure, whereas the difference between a theoretical (blue) and plasmid-derived sequences (red) indicates a selective pressure against this particular mutated residue within one single VR library, or within a context of all mutated VRs in the combined viral library (green). Because the sample of the sequenced plasmid library included only 21 individually sequenced random clones, due to the low sampling representation, the number of observed mutants (red) sometimes exceeds the expected frequency (blue) which always (with the exception of residues 444, 500, and 588) includes wt AAV2 residue.
  • FIG. 5A , FIG. 5B , FIG. 5C , FIG. 5D , FIG. 5E , and FIG. 5F show the selection of new variants from murine liver.
  • FIG. 5A Amino acid sequences of the variable regions (VRs) of the variants selected from the library after three rounds of directed evolution in the mouse liver. wt AAV2 sequences are shown in bold for comparison;
  • FIG. 5B Expression of luciferase transgene in the liver of mice at day 40 after tail-vein administration of rAAV vectors packaged into capsids as indicated;
  • FIG. 5C Time course of rAAV-Luc expression in the liver as assessed by Luciferase imaging in vivo.
  • FIG. 6 shows the nucleotide sequence of the Variable Region VIII (VR-VIII) in CapLib-3A and CapLib-3B plasmid and viral libraries. Shown are raw sequencing data to illustrate the selection pressure upon individual amino acid residues within one VR (VR-VIII) when all mutated VRs are combined in one library.
  • the mutagenized and wt AAV2 reference sequence are shown below;
  • FIG. 7 shows the alignment of mutated regions in consensus and sequences found in more than 4 copies. Amino acid substitutions are highlighted, those occurring in the consensus (top sequence) are in yellow. The 9 sequences under the consensus were those found in more than 4 copies; copy number is indicated on the left. The total number of mutations is shown to the right of each sequence;
  • FIG. 8 shows the amino acid composition in variable positions.
  • the X-axis designates residue position (VP1 numbering), and the Y-axis—the relative frequency of each amino acid at that position.
  • Amino acid color-coding is shown to the right of the graph. The respective VR boundaries are shown below amino acid numbers for better orientation;
  • FIG. 9 shows the comparison of theoretical and experimental complexity at each variable amino acid position.
  • the X-axis designates residue position (VP1 numbering), and the Y-axis—the theoretical (blue) and experimental (red) complexities at that position as reflected by total number of mutations.
  • the respective VR boundaries are shown below amino acid numbers for better orientation;
  • FIG. 10 shows a diagram of in vivo directed evolution.
  • Viral library is injected intravenously (via tail vein). Three days post-injection, the liver is harvested and subjected to collagenase/protease treatment. Episomal Hirt DNA was isolated from the whole organ and utilized to amplify AAV capsid gene sequences. A new, Round one-enriched library was prepared and the cycle was repeated. After 3 cycles, individual clones were sequenced and analyzed. The recurrent variants were selected and cap gene sequence was subcloned to derive a helper plasmid. rAAV vectors incorporating reporter genes were then packaged and their biodistribution was analyzed after intravenous administration;
  • FIG. 11 shows barcoded AAV cassette.
  • Top diagram of barcoded AAV transgene cassette: TR—AAV2 terminal repeat; CBA—CMV- ⁇ -actin promoter; luc—firefly luciferase cDNA; furin-2A—furin cleavage site 41 followed by a foot-and-mouth disease virus 2A ribosomal skip peptide 42 ; pA—bovine growth hormone gene polyadenylation site.
  • Bottom list of available 6 nt identifiers (barcodes); and
  • FIG. 12 shows a diagram of Fragment 3 assembly. Top strand scaffold oligonucleotides (shown in black color) were annealed to bottom strand anchor (shown in red) oligonucleotides. Degenerate positions (IUPAC nucleotide code) are highlighted by yellow background.
  • the present invention also provides improved rAAV-based genetic constructs that encode one or more therapeutic agents useful in the preparation of medicaments for the prevention, treatment, and/or amelioration of one or more diseases, disorders or dysfunctions resulting from a deficiency in one or more cellular components.
  • the invention provides libraries of rAAV-based genetic constructs encoding one or more selected molecules of interest, such as, for example, one or more diagnostic or therapeutic agents (including, e.g., proteins, polypeptides, peptides, antibodies, antigen binding fragments, siRNAs, RNAis, antisense oligo- and poly-nucleotides, ribozymes, and variants and/or active fragments thereof), for use in the diagnosis, prevention, treatment, and/or amelioration of symptoms of mammalian diseases, disorders, dysfunctions, deficiencies, defects, trauma, injury, and such like.
  • diagnostic or therapeutic agents including, e.g., proteins, polypeptides, peptides, antibodies, antigen binding fragments, siRNAs, RNAis, antisense oligo- and poly-nucleotides, ribozymes, and variants and/or active fragments thereof.
  • the present invention also provides infectious rAAV virions, as well as nucleic acid molecules and rAAV vectors that encode the novel AAV vectors described herein, as well as nucleic acids encoding one or more selected diagnostic and/or therapeutic agents for delivery to a selected population of mammalian cells.
  • the novel rAAV vectors, express constructs, and infectious virions and viral particles comprising them as disclosed herein preferably have an improved efficiency in transducing one or more of a variety of cells, tissues and organs of interest, when compared to wild-type, unmodified, expression constructs, and to the corresponding rAAV vectors and virions comprising them.
  • the improved rAAV vectors provided herein may transduce one or more selected host cells at higher-efficiencies (and often much higher efficiencies) than conventional, wild type (i.e., “unmodified”) rAAV vectors.
  • vectors prepared as described herein may be of different AAV serotypes, and the mutation of one or more of the sequences described herein may result in improved viral vectors, which are capable of higher-efficiency transduction than that of the corresponding, non-substituted vectors from which the mutants were prepared.
  • next-generation rAAV viral vectors may dramatically reduce the number of viral particles needed for a conventional gene therapy regimen.
  • the rAAV vectors prepared as described herein may be more stable, less immunogenic, and/or can be produced at much lower cost, or in a higher titer, than an equivalent wild type viral vector prepared in conventional fashion.
  • native amino acids normally present in the sequence of a viral capsid protein may be substituted by one or more non-native amino acids, including, a substitution of one or more amino acids not normally present at a particular residue in the corresponding wild-type protein.
  • the invention also provides isolated and purified polynucleotides that encode one or more of the disclosed viral vectors as described herein, as well as polynucleotides that encode such vectors.
  • the vector constructs of the present invention further include at least one nucleic acid segment that encodes a diagnostic or therapeutic molecule operably linked to a promoter capable of expressing the nucleic acid segment in a suitable host cell comprising the vector.
  • the transduction efficiency of a mutated rAAV vector will be higher than that of the corresponding, unmodified, wild-type vector, and as such, will preferably possess a transduction efficiency in a mammalian cell that is at least 2-fold, at least about 4-fold, at least about 6-fold, at least about 8-fold, at least about 10-fold, or at least about 12-fold or higher in a selected mammalian host cell than that of a virion that comprises a corresponding, unmodified, rAAV vector.
  • the transduction efficiency of the rAAV vectors provided herein will be at least about 15-fold higher, at least about 20-fold higher, at least about 25-fold higher, at least about 30-fold higher, or at least about 40, 45, or 50-fold or more greater than that of a virion that comprises a corresponding, wild-type vectors.
  • the present invention also concerns rAAV vectors, wherein the nucleic acid segment further comprises a promoter, an enhancer, a post-transcriptional regulatory sequence, a polyadenylation signal, or any combination thereof, operably linked to the nucleic acid segment that encodes the selected polynucleotide of interest.
  • the promoter is a heterologous promoter, a tissue-specific promoter, a cell-specific promoter, a constitutive promoter, an inducible promoter, or any combination thereof.
  • nucleic acid segments cloned into one or more of the novel rAAV expression vectors described herein will preferably express or encode one or more polypeptides, peptides, ribozymes, peptide nucleic acids, siRNAs, RNAis, antisense oligonucleotides, antisense polynucleotides, antibodies, antigen binding fragments, or any combination thereof.
  • the therapeutic agents useful in the invention may include one or more agonists, antagonists, anti-apoptosis factors, inhibitors, receptors, cytokines, cytotoxins, erythropoietic agents, glycoproteins, growth factors, growth factor receptors, hormones, hormone receptors, interferons, interleukins, interleukin receptors, nerve growth factors, neuroactive peptides, neuroactive peptide receptors, proteases, protease inhibitors, protein decarboxylases, protein kinases, protein kinase inhibitors, enzymes, receptor binding proteins, transport proteins or one or more inhibitors thereof, serotonin receptors, or one or more uptake inhibitors thereof, serpins, serpin receptors, tumor suppressors, diagnostic molecules, chemotherapeutic agents, cytotoxins, or any combination thereof.
  • vectors may also be prepared and packaged within virions of any known AAV serotype, including, for examples, AAV serotype 1 (AAV1), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), AAV serotype 6 (AAV6), AAV serotype 7 (AAV7), AAV serotype 8 (AAV8), AAV serotype 9 (AAV9), AAV serotype 10 (AAV10), AAV serotype 11 (AAV 11), or AAV serotype 12 (AAV12).
  • AAV serotype 1 AAV1
  • AAV3 AAV serotype 3
  • AAV4 AAV serotype 4
  • AAV serotype 5 AAV5
  • AAV serotype 6 AAV6
  • AAV serotype 7 AAV7
  • AAV8 AAV serotype 8
  • AAV9 AAV serotype 9
  • AAV9 AAV serotype 10
  • AAV10 AAV
  • the invention further provides populations and pluralities of such rAAV vectors as prepared herein, as well as virions, infectious viral particles, and mammalian host cells that include one or more nucleic acid segments encoding them.
  • the mammalian host cells will be human host cells, including, for example blood cells, stem cells, hematopoietic cells, CD34 + cells, liver cells, cancer cells, vascular cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dendritic cells, fibroblasts, or any other cell of mammalian origin, including, without limitation, hepatic (i.e., liver) cells, lung cells, cardiac cells, pancreatic cells, intestinal cells, diaphragmatic cells, renal (i.e., kidney) cells, neural cells, blood cells, bone marrow cells, or any one or more selected tissues of a mammal for which viral-based gene therapy is contemplated.
  • human host cells including, for example blood cells, stem cells, hematopoietic cells, CD34 + cells, liver cells, cancer cells, vascular cells, pancreatic cells, neural cells, ocular or retinal cells, epithelial or endothelial cells, dend
  • compositions that include one or more of the proteins nucleic acid segments viral vectors, host cells, or viral particles of the present invention together with one or more pharmaceutically-acceptable buffers, diluents, or excipients.
  • Such compositions may be included in one or more diagnostic or therapeutic kits, for diagnosing, preventing, treating or ameliorating one or more symptoms of a mammalian disease, injury, disorder, trauma or dysfunction.
  • the invention further includes a method for providing a mammal in need thereof with a diagnostically- or therapeutically-effective amount of a selected biological molecule, the method comprising providing to a cell, tissue or organ of a mammal in need thereof, an amount of an rAAV vector; and for a time effective to provide the mammal with a diagnostically- or a therapeutically-effective amount of the selected biological molecule.
  • the invention further provides a method for diagnosing, preventing, treating, or ameliorating at least one or more symptoms of a disease, a disorder, a dysfunction, an injury, an abnormal condition, or trauma in a mammal.
  • the method includes at least the step of administering to a mammal in need thereof one or more of the disclosed rAAV vectors, in an amount and for a time sufficient to diagnose, prevent, treat or ameliorate the one or more symptoms of the disease, disorder, dysfunction, injury, abnormal condition, or trauma in the mammal.
  • the invention also provides a method of transducing a population of mammalian cells.
  • the method includes at least the step of introducing into one or more cells of the population, a composition that comprises an effective amount of one or more of the rAAV vectors disclosed herein.
  • the invention also provides isolated nucleic acid segments that encode one or more of the mutant viral capsid proteins as described herein, and provides recombinant vectors, virus particles, infectious virions, and isolated host cells that comprise one or more of the improved vector sequences described and tested herein.
  • compositions as well as therapeutic and/or diagnostic kits that include one or more of the disclosed AAV compositions, formulated with one or more additional ingredients, or prepared with one or more instructions for their use.
  • the invention also demonstrates methods for making, as well as methods of using the disclosed improved rAAV vectors in a variety of ways, including, for example, ex situ, in vitro and in vivo applications, methodologies, diagnostic procedures, and/or gene therapy regimens. Because many of the improved vectors described herein are also resistant to proteasomal degradation, they possess significantly increased transduction efficiencies in vivo making them particularly well suited for viral vector-based human gene therapy regimens, and in particular, for delivering one or more genetic constructs to selected mammalian cells in vivo and/or in vitro.
  • the invention provides compositions comprising AAV vectors, virions, viral particles, and pharmaceutical formulations thereof, useful in methods for delivering genetic material encoding one or more beneficial or therapeutic product(s) to mammalian cells and tissues.
  • compositions and methods of the invention provide a significant advancement in the art through their use in the treatment, prevention, and/or amelioration of symptoms of one or more mammalian diseases. It is contemplated that human gene therapy will particularly benefit from the present teachings by providing new and improved viral vector constructs for use in the treatment of a number of diverse diseases, disorders, and dysfunctions.
  • the invention concerns libraries of rAAV vector mutants that demonstrate improved properties useful in the delivery of one or more therapeutic agents to selected mammalian cells, and particularly for use in the prevention, treatment, and/or amelioration of one or more disorders in a mammal into which the vector construct may be introduced.
  • the rAAV vectors of the present invention may optionally further include one or more enhancer sequences that are each operably linked to the nucleic acid segment.
  • exemplary enhancer sequences include, but are not limited to, one or more selected from the group consisting of a CMV enhancer, a synthetic enhancer, a liver-specific enhancer, an vascular-specific enhancer, a brain-specific enhancer, a neural cell-specific enhancer, a lung-specific enhancer, a muscle-specific enhancer, a kidney-specific enhancer, a pancreas-specific enhancer, and an islet cell-specific enhancer.
  • promoters useful in the practice of the invention include, without limitation, one or more heterologous, tissue-specific, constitutive or inducible promoters, including, for example, but not limited to, a promoter selected from the group consisting of a CMV promoter, a ⁇ -actin promoter, an insulin promoter, an enolase promoter, a BDNF promoter, an NGF promoter, an EGF promoter, a growth factor promoter, an axon-specific promoter, a dendrite-specific promoter, a brain-specific promoter, a hippocampal-specific promoter, a kidney-specific promoter, an elafin promoter, a cytokine promoter, an interferon promoter, a growth factor promoter, an ⁇ 1 -antitrypsin promoter, a brain cell-specific promoter, a neural cell-specific promoter, a central nervous system cell-specific promoter, a peripheral nervous system cell-specific promoter, an interleukin promoter
  • the vector-encoding nucleic acid segments may also further include one or more post-transcriptional regulatory sequences or one or more polyadenylation signals, including, for example, but not limited to, a woodchuck hepatitis virus post-transcription regulatory element, a polyadenylation signal sequence, or any combination thereof.
  • Exemplary diagnostic or therapeutic agents deliverable to host cells by the present vector constructs include, but are not limited to, an agent selected from the group consisting of a polypeptide, a peptide, an antibody, an antigen binding fragment, a ribozyme, a peptide nucleic acid, a siRNA, an RNAi, an antisense oligonucleotide, an antisense polynucleotide, and any combination thereof.
  • the rAAV vectors obtained by the disclosed methods will preferably encode at least one diagnostic or therapeutic protein or polypeptide selected from the group consisting of a molecular marker, an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-
  • the rAAV vectors of the present invention may include one or more nucleic acid segments that encode a polypeptide selected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, TGF-B2, TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(I87A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and any combination thereof.
  • the invention concerns genetically-modified, improved-transduction-efficiency rAAV vectors that include at least a first nucleic acid segment that encodes one or more therapeutic agents that alter, inhibit, reduce, prevent, eliminate, or impair the activity of one or more endogenous biological processes in the cell.
  • therapeutic agents may be those that selectively inhibit or reduce the effects of one or more metabolic processes, dysfunctions, disorders, or diseases.
  • the defect may be caused by injury or trauma to the mammal for which treatment is desired.
  • the defect may be caused the over-expression of an endogenous biological compound, while in other embodiments still; the defect may be caused by the under-expression or even lack of one or more endogenous biological compounds.
  • the genetically-modified rAAV vectors and expression systems of the present invention may also further optionally include a second distinct nucleic acid segment that comprises, consists essentially of, or consists of, one or more enhancers, one or more regulatory elements, one or more transcriptional elements, or any combination thereof, that alter, improve, regulate, and/or affect the transcription of the nucleotide sequence of interest expressed by the modified rAAV vectors.
  • the rAAV vectors of the present invention may further include a second nucleic acid segment that comprises, consists essentially of, or consists of, a CMV enhancer, a synthetic enhancer, a cell-specific enhancer, a tissue-specific enhancer, or any combination thereof.
  • the second nucleic acid segment may also further comprise, consist essentially of, or consist of, one or more intron sequences, one or more post-transcriptional regulatory elements, or any combination thereof.
  • the improved vectors and expression systems of the present invention may also optionally further include a polynucleotide that comprises, consists essentially of, or consists of, one or more polylinkers, restriction sites, and/or multiple cloning region(s) to facilitate insertion (cloning) of one or more selected genetic elements, genes of interest, or therapeutic or diagnostic constructs into the rAAV vector at a selected site within the vector.
  • a polynucleotide that comprises, consists essentially of, or consists of, one or more polylinkers, restriction sites, and/or multiple cloning region(s) to facilitate insertion (cloning) of one or more selected genetic elements, genes of interest, or therapeutic or diagnostic constructs into the rAAV vector at a selected site within the vector.
  • the exogenous polynucleotide(s) that may be delivered into suitable host cells by the improved, capsid-modified, rAAV vectors disclosed herein are preferably of mammalian origin, with polynucleotides encoding one or more polypeptides or peptides of human, non-human primate, porcine, bovine, ovine, feline, canine, equine, epine, caprine, or lupine origin being particularly preferred.
  • exogenous polynucleotide(s) that may be delivered into host cells by the disclosed capsid-modified viral vectors may, in certain embodiments, encode one or more proteins, one or more polypeptides, one or more peptides, one or more enzymes, or one or more antibodies (or antigen-binding fragments thereof), or alternatively, may express one or more siRNAs, ribozymes, antisense oligonucleotides, PNA molecules, or any combination thereof.
  • two or more different molecules may be produced from a single rAAV expression system, or alternatively, a selected host cell may be transfected with two or more unique rAAV expression systems, each of which may comprise one or more distinct polynucleotides that encode a therapeutic agent.
  • the invention also provides rAAV vector mutants that are comprised within an infectious adeno-associated viral particle or a virion, as well as pluralities of such virions or infectious particles.
  • Such vectors and virions may be comprised within one or more diluents, buffers, physiological solutions or pharmaceutical vehicles, or formulated for administration to a mammal in one or more diagnostic, therapeutic, and/or prophylactic regimens.
  • the vectors, virus particles, virions, and pluralities thereof of the present invention may also be provided in excipient formulations that are acceptable for veterinary administration to selected livestock, exotics, domesticated animals, and companion animals (including pets and such like), as well as to non-human primates, zoological or otherwise captive specimens, and such like.
  • the invention also concerns host cells that comprise at least one of the disclosed rAAV expression vectors, or one or more virus particles or virions that comprise such an expression vector.
  • host cells are particularly mammalian host cells, with human host cells being particularly highly preferred, and may be either isolated, in cell or tissue culture. In the case of genetically modified animal models, the transformed host cells may even be comprised within the body of a non-human animal itself.
  • the creation of recombinant non-human host cells, and/or isolated recombinant human host cells that comprise one or more of the disclosed rAAV vectors is also contemplated to be useful for a variety of diagnostic, and laboratory protocols, including, for example, means for the production of large-scale quantities of the rAAV vectors described herein.
  • virus production methods are particularly contemplated to be an improvement over existing methodologies including in particular, those that require very high titers of the viral stocks in order to be useful as a gene therapy tool.
  • the inventors contemplate that one very significant advantage of the present methods will be the ability to utilize lower titers of viral particles in mammalian transduction protocols, yet still retain transfection rates at a suitable level.
  • compositions comprising one or more of the disclosed rAAV vectors, expression systems, infectious AAV particles, or host cells also form part of the present invention, and particularly those compositions that further comprise at least a first pharmaceutically-acceptable excipient for use in therapy, and for use in the manufacture of medicaments for the treatment of one or more mammalian diseases, disorders, dysfunctions, or trauma.
  • Such pharmaceutical compositions may optionally further comprise one or more diluents, buffers, liposomes, a lipid, a lipid complex; or the tyrosine-modified rAAV vectors may be comprised within a microsphere or a nanoparticle.
  • compositions suitable for intramuscular, intravenous, or direct injection into an organ or tissue or a plurality of cells or tissues of a human or other mammal are particularly preferred, however, the compositions disclosed herein may also find utility in administration to discreet areas of the mammalian body, including for example, formulations that are suitable for direct injection into one or more organs, tissues, or cell types in the body.
  • Such injection sites include, but are not limited to, the brain, a joint or joint capsule, a synovium or subsynovium tissue, tendons, ligaments, cartilages, bone, peri-articular muscle or an articular space of a mammalian joint, as well as direct administration to an organ such as the heart, liver, lung, pancreas, intestine, brain, bladder, kidney, or other site within the patient's body, including, for example , introduction of the viral vectors via intraabdominal, intrathorascic, intravascular, or intracerebroventricular delivery.
  • aspects of the invention concern recombinant adeno-associated virus virion particles, compositions, and host cells that comprise, consist essentially of, or consist of, one or more of the rAAV vectors disclosed herein, such as for example pharmaceutical formulations of the vectors intended for administration to a mammal through suitable means, such as, by intramuscular, intravenous, intra-articular, or direct injection to one or more cells, tissues, or organs of a selected mammal.
  • compositions may be formulated with pharmaceutically-acceptable excipients as described hereinbelow, and may comprise one or more liposomes, lipids, lipid complexes, microspheres or nanoparticle formulations to facilitate administration to the selected organs, tissues, and cells for which therapy is desired.
  • Kits comprising one or more of the disclosed rAAV vectors (as well as one or more virions, viral particles, transformed host cells or pharmaceutical compositions comprising such vectors); and instructions for using such kits in one or more therapeutic, diagnostic, and/or prophylactic clinical embodiments are also provided by the present invention.
  • kits may further comprise one or more reagents, restriction enzymes, peptides, therapeutics, pharmaceutical compounds, or means for delivery of the composition(s) to host cells, or to an animal (e.g., syringes, injectables, and the like).
  • kits include those for treating, preventing, or ameliorating the symptoms of a disease, deficiency, dysfunction, and/or injury, or may include components for the large-scale production of the viral vectors themselves, such as for commercial sale, or for use by others, including e.g., virologists, medical professionals, and the like.
  • Another important aspect of the present invention concerns methods of use of the disclosed rAAV vectors, virions, expression systems, compositions, and host cells described herein in the preparation of medicaments for diagnosing, preventing, treating or ameliorating at least one or more symptoms of a disease, a dysfunction, a disorder, an abnormal condition, a deficiency, injury, or trauma in an animal, and in particular, in a vertebrate mammal.
  • Such methods generally involve administration to a mammal in need thereof, one or more of the disclosed vectors, virions, viral particles, host cells, compositions, or pluralities thereof, in an amount and for a time sufficient to diagnose, prevent, treat, or lessen one or more symptoms of such a disease, dysfunction, disorder, abnormal condition, deficiency, injury, or trauma in the affected animal.
  • the methods may also encompass prophylactic treatment of animals suspected of having such conditions, or administration of such compositions to those animals at risk for developing such conditions either following diagnosis, or prior to the onset of symptoms.
  • the exogenous polynucleotide will preferably encode one or more proteins, polypeptides, peptides, ribozymes, or antisense oligonucleotides, or a combination of these.
  • the exogenous polynucleotide may encode two or more such molecules, or a plurality of such molecules as may be desired.
  • two or more different molecules may be produced from a single rAAV expression system, or alternatively, a selected host cell may be transfected with two or more unique rAAV expression systems, each of which will provide unique heterologous polynucleotides encoding at least two different such molecules.
  • compositions comprising one or more of the disclosed rAAV vectors, expression systems, infectious AAV particles, host cells also form part of the present invention, and particularly those compositions that further comprise at least a first pharmaceutically-acceptable excipient for use in the manufacture of medicaments and methods involving therapeutic administration of such rAAV vectors.
  • Such pharmaceutical compositions may optionally further comprise liposomes, a lipid, a lipid complex; or the rAAV vectors may be comprised within a microsphere or a nanoparticle.
  • Pharmaceutical formulations suitable for intramuscular, intravenous, or direct injection into an organ or tissue of a human are particularly preferred.
  • kits for diagnosing, preventing, treating or ameliorating one or more symptoms of a mammalian disease, injury, disorder, trauma or dysfunction.
  • kits may be useful in the diagnosis, prophylaxis, and/or therapy or a human disease, and may be particularly useful in the treatment, prevention, and/or amelioration of one or more symptoms of cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disorder, neuromotor deficit, neuroskeletal impairment, neurological disability, neurosensory dysfunction, stroke, ischemia, ⁇ 1 -antitrypsin (AAT) deficiency, Batten's disease, Alzheimer's disease, sickle cell disease, ⁇ -thalassamia, Huntington's disease, Parkinson's disease, skeletal disease, trauma, pulmonary disease in a human.
  • AAT ⁇ 1 -antitrypsin
  • the invention also provides for the use of a composition disclosed herein in the manufacture of a medicament for treating, preventing or ameliorating the symptoms of a disease, disorder, dysfunction, injury or trauma, including, but not limited to, the treatment, prevention, and/or prophylaxis of a disease, disorder or dysfunction, and/or the amelioration of one or more symptoms of such a disease, disorder or dysfunction.
  • the invention also provides a method for treating or ameliorating the symptoms of such a disease, injury, disorder, or dysfunction in a mammal.
  • Such methods generally involve at least the step of administering to a mammal in need thereof, one or more of the rAAV vectors as disclosed herein, in an amount and for a time sufficient to treat or ameliorate the symptoms of such a disease, injury, disorder, or dysfunction in the mammal.
  • Such treatment regimens are particularly contemplated in human therapy, via administration of one or more compositions either intramuscularly, intravenously, subcutaneously, intrathecally, intraperitoneally, or by direct injection into an organ or a tissue of the mammal under care.
  • the invention also provides a method for providing to a mammal in need thereof, a therapeutically-effective amount of an rAAV composition of the present invention, in an amount, and for a time effective to provide the patient with a therapeutically-effective amount of the desired therapeutic agent(s) encoded by one or more nucleic acid segments comprised within the rAAV vector.
  • exemplary therapeutic agents include, but are not limited to, a polypeptide, a peptide, an antibody, an antigen-binding fragment, a ribozyme, a peptide nucleic acid, an siRNA, an RNAi, an antisense oligonucleotide, an antisense polynucleotide, or a combination thereof.
  • the genetic constructs of the present invention may be prepared in a variety of compositions, and may also be formulated in appropriate pharmaceutical vehicles for administration to human or animal subjects.
  • the invention also provides compositions comprising one or more of the disclosed rAAV vectors, expression systems, virions, viral particles, mammalian cells, or combinations thereof.
  • the present invention provides pharmaceutical formulations of one or more rAAV vectors disclosed herein for administration to a cell or an animal, either alone or in combination with one or more other modalities of therapy, and in particular, for therapy of human cells, tissues, and diseases affecting man.
  • Formulation of pharmaceutically-acceptable excipients and carrier solutions is well-known to those of skill in the art, as is the development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., oral, parenteral, intravenous, intranasal, intra-articular, intramuscular administration and formulation.
  • carrier is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s) or such like, or a combination thereof, that is pharmaceutically acceptable for administration to the relevant animal.
  • delivery vehicles for chemical compounds in general, and chemotherapeutics in particular is well known to those of ordinary skill in the pharmaceutical arts. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the diagnostic, prophylactic, and therapeutic compositions is contemplated.
  • One or more supplementary active ingredient(s) may also be incorporated into, or administered in association with, one or more of the disclosed chemotherapeutic compositions.
  • chimeric rcAAV refers to a replication-competent AAV-derived nucleic acid containing at least one nucleotide sequence that 1) encodes an AAV protein and 2) differs from the corresponding native nucleotide sequence in one or more bases.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • an effective amount would be understood by those of ordinary skill in the art to provide a therapeutic, prophylactic, or otherwise beneficial effect against the organism, its infection, or the symptoms of the organism or its infection, or any combination thereof
  • expression control sequence refers to any genetic element (e.g., polynucleotide sequence) that can exert a regulatory effect on the replication or expression (transcription or translation) of another genetic element.
  • Common expression control sequences include promoters, polyadenylation (polyA) signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites (IRES), enhancers, and the like.
  • a “tissue specific expression control sequence” is one that exerts a regulatory effect on the replication or expression (transcription or translation) of another genetic element in only one type of tissue or a small subset of tissues.
  • helper function is meant as a functional activity performed by a nucleic acid or polypeptide that is derived from a virus such as Adenovirus (Ad) or herpesvirus and that facilitates AAV replication in a host cell.
  • Ad Adenovirus
  • herpesvirus a virus that facilitates AAV replication in a host cell.
  • heterologous is defined in relation to a predetermined referenced gene sequence.
  • a heterologous promoter is defined as a promoter which does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation.
  • a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
  • the term “homology” refers to a degree of complementarity between two or more polynucleotide or polypeptide sequences.
  • the word “identity” may substitute for the word “homology” when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence.
  • Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.
  • homologous means, when referring to polynucleotides, sequences that have the same essential nucleotide sequence, despite arising from different origins. Typically, homologous nucleic acid sequences are derived from closely related genes or organisms possessing one or more substantially similar genomic sequences. By contrast, an “analogous” polynucleotide is one that shares the same function with a polynucleotide from a different species or organism, but may have a significantly different primary nucleotide sequence that encodes one or more proteins or polypeptides that accomplish similar functions or possess similar biological activity. Analogous polynucleotides may often be derived from two or more organisms that are not closely related (e.g., either genetically or phylogenetically).
  • nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (or other algorithms available to persons of ordinary skill) or by visual inspection.
  • the phrase “in need of treatment” refers to a judgment made by a caregiver such as a physician or veterinarian that a patient requires (or will benefit in one or more ways) from treatment. Such judgment may made based on a variety of factors that are in the realm of a caregiver's expertise, and may include the knowledge that the patient is ill as the result of a disease state that is treatable by one or more compound or pharmaceutical compositions such as those set forth herein.
  • isolated or “biologically pure” refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.
  • isolated polynucleotides in accordance with the invention preferably do not contain materials normally associated with those polynucleotides in their natural, or in situ, environment.
  • kit may be used to describe variations of the portable, self-contained enclosure that includes at least one set of components to conduct one or more of the diagnostic or therapeutic methods of the invention.
  • a vector library refers to a collection of elements that differ from one another in at least one aspect.
  • a vector library is a collection of at least two vectors that differ from one another by at least one nucleotide.
  • a “virion library” is a collection of at least two virions that differ from one another by at least one nucleotide or at least one capsid protein.
  • Link refers to any method known in the art for functionally connecting one or more proteins, peptides, nucleic acids, or polynucleotides, including, without limitation, recombinant fusion, covalent bonding, disulfide bonding, ionic bonding, hydrogen bonding, electrostatic bonding, and the like.
  • master library refers to a pool of rAAV virions composed of chimeric rcAAV vectors encapsidated in cognate chimeric capsids (e.g., capsids containing a degenerate or otherwise modified Cap protein).
  • nucleic acid molecule or polypeptide when referring to a nucleic acid molecule or polypeptide, the term “native” refers to a naturally-occurring (e.g., a WT) nucleic acid or polypeptide.
  • naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring.
  • laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally occurring animals
  • nucleic acid means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • the phrases “cap nucleic acid,” “cap gene,” and “capsid gene” as used herein mean a nucleic acid that encodes a Cap protein.
  • Examples of cap nucleic acids include “wild-type” (WT) Cap-encoding nucleic acid sequences from AAV serotypes 1, 2, and 5; a native form cap cDNA; a nucleic acid having sequences from which a cap cDNA can be transcribed; and/or allelic variants and homologs of the foregoing.
  • WT wild-type
  • the term “patient” refers to any host that can receive one or more of the pharmaceutical compositions disclosed herein.
  • the subject is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
  • a “patient” refers to any animal host including without limitation any mammalian host.
  • the term refers to any mammalian host, the latter including but not limited to, human and non-human primates, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, ranines, racines, vulpines, and the like, including livestock, zoological specimens, exotics, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
  • phrases “pharmaceutically-acceptable” refers to molecular entities and compositions that preferably do not produce an allergic or similar untoward reaction when administered to a mammal, and in particular, when administered to a human.
  • pharmaceutically acceptable salt refers to a salt that preferably retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects.
  • salts include, without limitation, acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, and the like); and salts formed with organic acids including, without limitation, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic (embonic) acid, alginic acid, naphthoic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; salts with polyvalent metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; salts formed with an organic cation formed from N,N′-dibenzylethylenedi
  • pharmaceutically acceptable salt refers to a compound of the present disclosure derived from pharmaceutically acceptable bases, inorganic or organic acids.
  • suitable acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, trifluoroacetic and benzenesulfonic acids.
  • Salts derived from appropriate bases include, but are not limited to, alkalis such as sodium and ammonia.
  • Plasmids and vectors of the present invention may include one or more genetic elements as described herein arranged such that an inserted coding sequence can be transcribed and translated in a suitable expression cells.
  • the plasmid or vector may include one or more nucleic acid segments, genes, promoters, enhancers, activators, multiple cloning regions, or any combination thereof, including segments that are obtained from or derived from one or more natural and/or artificial sources.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
  • polypeptide terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
  • the term further includes polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • the terms “prevent,” “preventing,” “prevention,” “suppress,” “suppressing,” and “suppression” as used herein refer to administering a compound either alone or as contained in a pharmaceutical composition prior to the onset of clinical symptoms of a disease state so as to prevent any symptom, aspect or characteristic of the disease state. Such preventing and suppressing need not be absolute to be deemed medically useful.
  • promoter refers to a region or regions of a nucleic acid sequence that regulates transcription.
  • Protein is used herein interchangeably with “peptide” and “polypeptide,” and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject.
  • polypeptide is preferably intended to refer to any amino acid chain length, including those of short peptides from two to about 20 amino acid residues in length, oligopeptides from about 10 to about 100 amino acid residues in length, and longer polypeptides including those of about 100 or more amino acid residues in length.
  • polypeptides and proteins of the present invention also include polypeptides and proteins that are or have been post-translationally modified, and include any sugar or other derivative(s) or conjugate(s) added to the backbone amino acid chain.
  • proliferative typed is meant a nucleic acid or genome derived from a first AAV serotype that is encapsidated (packaged) into an AAV capsid containing at least one AAV Cap protein of a second serotype differing from the first serotype.
  • rcAAV vector refers to a replication-competent AAV-derived nucleic acid capable of DNA replication in a cell without any additional AAV genes or gene products.
  • recombinant indicates that the material (e.g., a polynucleotide or a polypeptide) has been artificially or synthetically (non-naturally) altered by human intervention. The alteration can be performed on the material within or removed from, its natural environment or state. Specifically, e.g., a promoter sequence is “recombinant” when it is produced by the expression of a nucleic acid segment engineered by the hand of man.
  • a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other procedures, or by chemical or other mutagenesis
  • a “recombinant polypeptide” or “recombinant protein” is a polypeptide or protein which is produced by expression of a recombinant nucleic acid
  • a “recombinant virus,” e.g., a recombinant AAV virus is produced by the expression of a recombinant nucleic acid.
  • regulatory element refers to a region or regions of a nucleic acid sequence that regulates transcription.
  • exemplary regulatory elements include, but are not limited to, enhancers, post-transcriptional elements, transcriptional control sequences, and such like.
  • RNA segment refers to an RNA molecule that has been isolated free of total cellular RNA of a particular species. Therefore, RNA segments can refer to one or more RNA segments (either of native or synthetic origin) that have been isolated away from, or purified free from, other RNAs. Included within the term “RNA segment,” are RNA segments and smaller fragments of such segments.
  • seed library is meant a pool of AAV virions composed of chimeric rcAAV vectors encapsidated into AAV capsids of a single serotype.
  • substantially corresponds to denote a characteristic of a nucleic acid or an amino acid sequence, wherein a selected nucleic acid or amino acid sequence has at least about 70 or about 75 percent sequence identity as compared to a selected reference nucleic acid or amino acid sequence. More typically, the selected sequence and the reference sequence will have at least about 76, 77, 78, 79, 80, 81, 82, 83, 84 or even 85 percent sequence identity, and more preferably, at least about 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95 percent sequence identity. More preferably still, highly homologous sequences often share greater than at least about 96, 97, 98, or 99 percent sequence identity between the selected sequence and the reference sequence to which it was compared.
  • the percentage of sequence identity may be calculated over the entire length of the sequences to be compared, or may be calculated by excluding small deletions or additions which total less than about 25 percent or so of the chosen reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome.
  • the reference sequence will typically comprise at least about 18-25 nucleotides, more typically at least about 26 to 35 nucleotides, and even more typically at least about 40, 50, 60, 70, 80, 90, or even 100 or so nucleotides.
  • the extent of percent identity between the two sequences will be at least about 80%, preferably at least about 85%, and more preferably about 90% or 95% or higher, as readily determined by one or more of the sequence comparison algorithms well-known to those of skill in the art, such as e.g., the FASTA program analysis described by Pearson and Lipman (1988).
  • structural gene is intended to generally describe a polynucleotide, such as a gene, that is expressed to produce an encoded peptide, polypeptide, protein, ribozyme, catalytic RNA molecule, or antisense molecule.
  • subject describes an organism, including a mammal such as a human primate, to which treatment with one or more of the disclosed compositions may be provided.
  • Mammalian species that may benefit from the disclosed treatment methods include, without limitation, humans, non-human primates such as apes; chimpanzees; monkeys, and orangutans, domesticated animals, including dogs and cats, as well as livestock such as horses, cattle, pigs, sheep, and goats, or other mammalian species including, without limitation, mice, rats, guinea pigs, rabbits, hamsters, and the like.
  • terminal repeat or “TR” mean a nucleic acid sequence derived from an AAV that is required in cis for replication and packaging of AAV.
  • Transcriptional regulatory element refers to a polynucleotide sequence that activates transcription alone or in combination with one or more other nucleic acid sequences.
  • a transcriptional regulatory element may include, for example, one or more promoters, one or more response elements, one or more negative regulatory elements, one or more enhancers, or any combination thereof.
  • transcription factor recognition site and a “transcription factor binding site” refer to a polynucleotide sequence(s) or sequence motif(s) that are identified as being sites for the sequence-specific interaction of one or more transcription factors, frequently taking the form of direct protein-DNA binding.
  • transcription factor binding sites can be identified by DNA footprinting, gel mobility shift assays, and the like, and/or can be predicted based on known consensus sequence motifs, or by other methods known to one of ordinary skill in the relevant molecular biological and virology arts.
  • Transcriptional unit refers to a polynucleotide sequence that comprises at least a first structural gene operably linked to at least a first cis-acting promoter sequence and optionally linked operably to one or more other cis-acting nucleic acid sequences necessary for efficient transcription of the structural gene sequences, and at least a first distal regulatory element as may be required for the appropriate tissue-specific and developmental transcription of the structural gene sequence operably positioned under the control of the promoter and/or enhancer elements, as well as any additional cis sequences that are necessary for efficient transcription and translation (e.g., polyadenylation site(s), mRNA stability controlling sequence(s), etc.
  • transformed cell is intended to mean a host cell whose nucleic acid complement has been altered by the introduction of one or more exogenous polynucleotides into that cell.
  • transformation is intended to generally describe a process of introducing an exogenous polynucleotide sequence (e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule) into a host cell or protoplast in which the exogenous polynucleotide is incorporated into at least a first chromosome or is capable of autonomous replication within the transformed host cell.
  • an exogenous polynucleotide sequence e.g., a viral vector, a plasmid, or a recombinant DNA or RNA molecule
  • Transfection, electroporation, and “naked” nucleic acid uptake all represent examples of techniques used to transform a host cell with one or more polynucleotides.
  • the terms “treat,” “treating,” and “treatment” refer to the administration of one or more compounds (either alone or as contained in one or more pharmaceutical compositions) after the onset of clinical symptoms of a disease state so as to reduce, or eliminate any symptom, aspect or characteristic of the disease state. Such treating need not be absolute to be deemed medically useful.
  • the terms “treatment,” “treat,” “treated,” or “treating” may refer to therapy, or to the amelioration or the reduction, in the extent or severity of disease, of one or more symptom thereof, whether before or after its development afflicts a patient.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked, e.g., a plasmid.
  • a vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked.
  • An “rAAV vector” is a recombinant AAV-derived nucleic acid containing at least one terminal repeat (TR) sequence.
  • virion is meant to describe a virus particle that contains a nucleic acid and a protein coat (capsid).
  • An “rAAV virion” is a virion that includes nucleic acid sequences and/or proteins derived from a rAAV vector.
  • sequence essentially as set forth in SEQ ID NO:X means that the sequence substantially corresponds to a portion of SEQ ID NO:X and has relatively few nucleotides (or amino acids in the case of polypeptide sequences) that are not identical to, or a biologically functional equivalent of, the nucleotides (or amino acids) of SEQ ID NO:X.
  • biologically functional equivalent is well understood in the art, and is further defined in detail herein.
  • sequences that have about 85% to about 90%; or more preferably, about 91% to about 95%; or even more preferably, about 96% to about 99%; of nucleotides that are identical or functionally equivalent to one or more of the nucleotide sequences provided herein are particularly contemplated to be useful in the practice of the invention.
  • Suitable standard hybridization conditions for the present invention include, for example, hybridization in 50% formamide, 5 ⁇ Denhardt's solution, 5 ⁇ SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/ml of denatured salmon sperm DNA at 42° C. for 16 h followed by 1 hr sequential washes with 0.1 ⁇ SSC, 0.1% SDS solution at 60° C. to remove the desired amount of background signal.
  • Lower stringency hybridization conditions for the present invention include, for example, hybridization in 35% formamide, 5 ⁇ Denhardt's solution, 5 ⁇ SSC, 25 mM sodium phosphate, 0.1% SDS and 100 ⁇ g/ml denatured salmon sperm DNA or E. coli DNA at 42° C. for 16 h followed by sequential washes with 0.8 ⁇ SSC, 0.1% SDS at 55° C.
  • Those of skill in the art will recognize that conditions can be readily adjusted to obtain the desired level of stringency.
  • nucleic acid segments that are complementary, essentially complementary, and/or substantially complementary to at least one or more of the specific nucleotide sequences specifically set forth herein.
  • Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules.
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to one or more of the specific nucleic acid segments disclosed herein under relatively stringent conditions such as those described immediately above.
  • the probes and primers of the present invention may be of any length.
  • an algorithm defining all probes or primers contained within a given sequence can be proposed:
  • n to n +y where n is an integer from 1 to the last number of the sequence and y is the length of the probe or primer minus one, where n +y does not exceed the last number of the sequence.
  • exemplary primer or probe sequence include, without limitation, sequences corresponding to bases 1 to 35, bases 2 to 36, bases 3 to 37, bases 4 to 38, and so on over the entire length of the sequence.
  • probes or primers may correspond to the nucleotides from the first basepair to by 40, from the second by of the sequence to by 41, from the third by to by 42, and so forth
  • probes or primers may correspond to a nucleotide sequence extending from by 1 to by 50, from by 2 to by 51, from by 3 to by 52, from by 4 to by 53, and so forth.
  • nucleic acid segments of the present invention in combination with an appropriate detectable marker (i.e., a “label,”), such as in the case of employing labeled polynucleotide probes in determining the presence of a given target sequence in a hybridization assay.
  • an appropriate detectable marker i.e., a “label,”
  • a wide variety of appropriate indicator compounds and compositions are known in the art for labeling oligonucleotide probes, including, without limitation, fluorescent, radioactive, enzymatic or other ligands, such as avidin/biotin, etc., which are capable of being detected in a suitable assay.
  • an enzyme tag such as urease, alkaline phosphatase or peroxidase
  • colorimetric, chromogenic, or fluorigenic indicator substrates are known that can be employed to provide a method for detecting the sample that is visible to the human eye, or by analytical methods such as scintigraphy, fluorimetry, spectrophotometry, and the like, to identify specific hybridization with samples containing one or more complementary or substantially complementary nucleic acid sequences.
  • multiplexing assays where two or more labeled probes are detected either simultaneously or sequentially, it may be desirable to label a first oligonucleotide probe with a first label having a first detection property or parameter (for example, an emission and/or excitation spectral maximum), which also labeled a second oligonucleotide probe with a second label having a second detection property or parameter that is different (i.e., discreet or discernable from the first label.
  • first detection property or parameter for example, an emission and/or excitation spectral maximum
  • polynucleotides, nucleic acid segments, nucleic acid sequences, and the like include, but are not limited to, DNAs (including and not limited to genomic or extragenomic DNAs), genes, peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
  • DNAs including and not limited to genomic or extragenomic DNAs
  • genes include peptide nucleic acids (PNAs) RNAs (including, but not limited to, rRNAs, mRNAs and tRNAs), nucleosides, and suitable nucleic acid segments either obtained from natural sources, chemically synthesized, modified, or otherwise prepared or synthesized in whole or in part by the hand of man.
  • PNAs peptide nucleic acids
  • Methodologies to improve existing AAV vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues.
  • both approaches were integrated in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified.
  • Individual sub-libraries were first assembled in order to pre-select compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants.
  • the successful families were interbred to derive a combined library of about 1 ⁇ 10 8 complexity.
  • Next-Gen sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution thus providing guidance for future designs.
  • the utility of the library in gene therapy applications by deriving an AAV2-based vector characterized by 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8.
  • CapLib-6 amino acid sequence of the AAV2 capsid showing altered positions in the library (X) is shown below:
  • the library was injected in the bloodstream of mice, and capsid DNA was amplified from the liver three days later and used to generate a new viral library. After three such rounds of in vivo selection, the following variants were obtained:
  • Loop I 444 IV V clones subst (SEQ ID NOS) AAV2 SQSGASN Y NTPSGTTTQSRLQFS TSADNNNSEYSWTGATKYH (SEQ ID NO: 106) LI-A SASGASN F NSEGGSLTQSSLGFS TDGENNNSDFSWTGATKYH 21 14 (SEQ ID NO: 107) LI-B SQSGASN Y NTPSGTTTQSRLQFS TDGENNNSDFSWTGATKYH 4 5 (SEQ ID NO: 108) LI-C SASGASN Y NTPSGTTTQSRLQFS TSADNNNSEFSWPGATTYH 3 4 (SEQ ID NO: 109) LI-D SQSGASN F NSEGGSLTQSSLGFS TDGENNNSDFSWTGATKYH 3 13 (SEQ ID NO: 110) LI-E SASGASN Y NTPSGSLTQSSLGFS TDGENNNSDFSWTGATKYH 2 10 (SEQ ID NO
  • AAV is a single-stranded DNA virus belonging to the Parvoviridae family (Muzyczka and Berns, 2001).
  • AAV-derived vectors are promising tools for human gene therapy applications because of their absence of pathogenicity, episomal localization and stable transgene expression (Wu et al., 2006).
  • significant limitations to the clinical use of AAV are its promiscuity and its susceptibility to neutralization by human antibodies (Louis-Jeune et al., 2013). Both of these limitations are determined by the nature of amino acid residues exposed at the surface of the capsid.
  • Two main strategies are generally employed to improve AAV vectors: 1) mutagenizing capsid residues to facilitate binding, entry, and/or intracellular trafficking through a rational approach based on the knowledge of virus biology (Wu et al., 2000; Zhong et al., 2008; Lochrie et al., 2005; Aslanidi et al., 2012; Li et al., 2012; Gabriel et al., 2013; Pandya et al., 2013; Sen et al., 2013; Aslanidi et al., 2013); and 2) utilizing a process of directed evolution, a high-throughput method of introducing molecular modifications into the AAV capsids, manipulating both diversity and selection in a vastly hastened emulation of natural evolution (Müller et al., 2003; Perabo et al., 2003; Maheshri et al., 2006; Michelfelder et al., 2007; Kwon and Schaffer, 2008; Li
  • the utility of the resulting library was demonstrated by enhancing the transduction of murine liver by 20-fold with an AAV2-derived vector containing only four mutations—a combination of rational mutagenesis and directed evolution.
  • Fragments were assembled by Enzymatic Ligation Assisted by Nucleases (ELAN) as described (Cost and Cozzarelli, 2007). Fragments for the six individual VR lineages were generated by overlap extension (Horton et al., 1989) of PCR products derived from pSub201 (Samulski et al., 1987) for the wild-type regions and from DHGFR products (or from the ELAN product if no suitable DHGFR template was available) for the variable regions.
  • ELAN Enzymatic Ligation Assisted by Nucleases
  • the assembled fragments were inserted into linearized pSubEagApa (a pSub201 derivative containing a deletion between ApaI sites at 3764 and 4049 and including an EagI site at position 4373, a silent mutation, thus allowing reconstitution of a full-length cap gene after insertion of a fragment between the ApaI and EagI sites), using isothermal DNA assembly (Gibson et al., 2009).
  • Viral libraries were generated as previously described (Maheshri et al., 2006) except that HEK 293 cells were co-transfected with 10 ng of the library plasmid and 70 ⁇ g of pHelper (a molar ratio of 1:5000) for each 15-cm dish.
  • AAV was purified using iodixanol gradient (Zolotukhin et al., 1999) and titered by qPCR using primers specific to the rep gene (forward: 5′-GCAAGACCGGATGTTCAAAT-3′ (SEQ ID NO:165), reverse: 5′-CCTCAACCACGTGATCCTTT-3′, (SEQ ID NO:166).
  • primers specific to the rep gene forward: 5′-GCAAGACCGGATGTTCAAAT-3′ (SEQ ID NO:165), reverse: 5′-CCTCAACCACGTGATCCTTT-3′, (SEQ ID NO:166).
  • overlapping fragments containing variable regions were amplified from purified DNA isolated from the individual viral libraries, and assembled using overlap extension.
  • the product was inserted into linearized pSubEagApa as described above.
  • the final viral library was generated, purified and quantified as described above.
  • the resulting amplification product was gel-purified. It was then processed at the UF ICBR NextGen core laboratory on a PacBio RS instrument using one SMRT cell. In order to analyze the data, dedicated code was written using the Python 2.7 programming language. The code interpreted the original fastq file containing the CCS reads, corrected reads by aligning them to a reference sequence, translated the corrected reads into protein sequences, and then analyzed the results. Despite a high occurrence of sequencing errors, useful reads could be recovered, because most of the capsid sequence is conserved, and only the sequences in the variable regions are needed.
  • variable regions included 182 nucleotides out of 1399.
  • the corrected reads generated by caplib were therefore derived from the CCS reads that likely had no errors in the variable regions (exact match with the reference sequence, including the ambiguous nucleotides). Obviously, this approach excludes all reads with possible insertions or deletions in the variable regions, and ignores any possible variation in the conserved regions.
  • amplification products were inserted into linearized pSubEagApa as described above. Liver-enriched libraries were then generated, purified and quantified as described above.
  • amplified capsid DNA was inserted into linearized pACG2-m56, a vector derived from pACG-2 (Li et al., 1997) with the same modifications as pSubEagApa (deletion between two ApaI sites and introduction of an EagI site allowing cloning of amplified capsid fragments between the ApaI and EagI sites). Random clones were then analyzed by sequencing.
  • HEK 293 cells were co-transfected with selected capsid variants in pACG2-m56, luciferase-expressing barcoded vector pTR-UF50-BC (GenBank accession # KF926476) and pHelper in equimolar amounts, in a separate transfection for each variant. Each capsid variant was therefore physically linked to a different sequence identifier (barcode). Resulting transgenic AAVs were purified and quantified as described above, except that primers specific to the CBA promoter were used for qPCR:
  • Barcode Sequencing Sequencing of barcoded DNA was performed at the UF ICBR NextGen core laboratory using an IonTorrent PGM system. Samples were amplified using the following barcoded primers:
  • N designates a unique sequence.
  • Each read therefore contains 3 barcodes: one identifying the capsid variant, located in the template, and two identifying the tissue sample, located in the primers. Sequencing data was analyzed by dna-barcode, a dedicated script written in Python 2.7.
  • hFIX Expression Viral vectors containing either AAV8 or LiC capsid and ApoE-hAAT-hF9 vector (Manno et al., 2006) were produced as described above. Male C57BL/6 mice (Jackson Laboratories), 6 to 8 weeks old, were injected in the tail vein with 1 ⁇ 10 10 vg of either preparation, 4 mice for each vector. Injected mice were followed for three months and bled monthly from the retro-orbital plexus using heparinized microcapillary tubes. Plasma levels of hFIX antigen were measured by ELISA as published (Mingozzi et al., 2003; Cao et al., 2009).
  • each variable region including nucleotide sequence and resulting amino acid diversity, is shown in the accompanying figures.
  • the AAV2 capsid gene fragment incorporating all substitutions was assembled from synthetic oligonucleotides and inserted into a plasmid vector containing the AAV2 genome from which the corresponding WT sequence had been removed.
  • the plasmid library with an estimated complexity of 1 x 10 8 , was analyzed by sequencing random clones and was found to reflect accurately the library's design, including expected nucleotide substitutions positions and types. This highly representative plasmid library, however, failed to produce packaged virus of higher titer.
  • An apparent explanation was the presence of an alternative open reading frame (ORF) coding for the assembly-activating protein (AAP) (Sonntag et al., 2010).
  • AAP ORF assembly-activating protein
  • AAP ORF assembly-activating protein
  • the resulting sequence includes 136 degenerate positions and encodes 59 variable amino acid positions, each with 2 to 16 permutations resulting in a theoretical complexity (total number of possible amino acid combinations) of 4 ⁇ 10 (Govindasamy et al., 2006).
  • Three-dimensional models of the VP3 monomer and the assembled capsid were rendered with variable amino acid positions colored according to VR identity.
  • a plasmid library was generated, with an estimated complexity of 9.2 ⁇ 10 7 , as deduced by extrapolating colony counts and correcting for positive clones. Its quality was assessed by sequencing a random sample of 21 clones where all corresponding 21 protein sequences were found to be unique. Each differed from the reference AAV2 sequence by 24 to 41 amino acid substitutions. Substitutions were distributed among five to seven VRs per sequence. VRs II and III were WT in all cases. Because of the assembly procedure, positions 444 and 500 exhibited Y/F polymorphism, with 1/21 and 8/21 containing WT Tyr residues in the respective positions.
  • Amino acid composition at each variable position was determined. Although some level of diversity is visible at most positions (only position 383 has a single identity), in many cases a single amino acid is predominant. However, 14 positions in which no single variant occurs in more than 50% of the sequences can be found, in VRs I (position 263), IV (450 to 461) and VII (548 to 550, 555 and 556).
  • Liver is an important gene therapy target to treat many inherited disorders (Sands, 2011; Sharland et al., 2010).
  • Several serotypes target liver preferentially, with the highest transduction efficiency shown by AAV8 (Gao et al., 2002; Nakai et al., 2004) in murine liver, with expression levels two orders of magnitude higher than those of AAV2.
  • AAV8 is dramatically lower (Hurlbut et al., 2010), although AAV8 is still being considered one of the best candidate vectors for human liver-directed gene therapy.
  • AAV2 was used as the library's backbone, we asked whether a novel capsid variant could be isolated targeting the liver with an efficiency comparable to that of AAV8.
  • Bold characters in motifs indicate amino acid substitutions (compared to the AAV2 reference).
  • the 2 columns on the right show the motif occurrences in the library sequencing sample (CapLib) and in the sequenced liver-selected variants.
  • Variant capsids LiA and LiC were packaged with luciferase-expressing barcoded vector pTR-UF50-BC.
  • the dynamics of luciferase expression in the liver were considerably different for all serotypes and variants: AAV8 mediated a fast initial increase followed by a slow leveling off. LiA followed a similar trend at a lower level of expression.
  • LiC on the other hand, provided steady gain converging with the levels of AAV8 after about 30 days. At day 40, the AAV8 and LiC-mediated luciferase expression exceeded that of AAV2 by 20-fold. Consistent with luciferase expression, hFIX transgene expression driven by ApoE-hAAT promoter followed the same pattern showing similar efficiencies for AAV8 and LiC, and a two-fold lower efficiency for LiA.
  • mice were euthanized 42 days after injection to examine biodistribution of viral genomes in various tissues and organs. Total DNA was isolated from samples, quantitated and used as template for qPCR to determine viral genome copy numbers.
  • a bioinformatic analysis of the existing sequence database of 150 AAV naturally-occurring variants was used to create a consensus AAV capsid template whereby nine variable regions incorporated alternative residues present in a given position in at least one of the serotypes.
  • This in silico-derived algorithm was converted into a combinatorial plasmid DNA library using the only feasible approach—de novo DNA synthesis. Even though the plasmid library was found to reflect faithfully the initial design, a new selection trait of 3D structural compatibility had to be introduced to isolate structurally sound variants within each individual VR. Subsequently, the successful families were interbred to derive a combined viral library of about 1 ⁇ 10 8 complexity, which was characterized in depth using Next-Gen sequencing.
  • the Q263A mutation is also one of the mutations in AAV2.5, the muscle tropic vector derived from AAV2 (Bowles et al., 2011).
  • the VR-V residues are within the ADK1 a footprint on AAV1 and the 4E4 footprint on AAV1 (Gurda et al., 2013).
  • Residues 490-500 are within the C37B footprint on AAV2 and 5H7 footprint on AAV1 (Gurda et al., 2013), and have been reported to affect liver tropism in AAV9 (Pu Norwayla et al., 2011).
  • VR-I is raised on the capsid and is not interacting with other monomers; VR-IV protrudes the furthest from the surface and only the base of this VR is interacting with other VP monomers.
  • VRs I, IV, VII, V and VI have the highest content of mutant positions, while VRs III, IX and, to a lesser extent, VIII are mostly invariable, consistent with previous reports (Ng et al., 2010).
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically and/or physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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