Viral vectors exhibiting improved gene delivery properties

Abstract

To provide viral vectors exhibiting improved gene delivery properties.SOLUTION: The technology described herein provides viral capsid polypeptides bearing mutations that alters tissue tropism of a virus comprising the viral capsid polypeptide. In various embodiments, tissue tropism to the heart, kidney, liver, lung, spleen or blood is altered. Not only the tropism of a viral vector can be improved by incorporating amino acid changes that increase tropism for a desired tissue, but nucleic acid delivery to a given tissue or cell type can also be improved by reducing the efficiency with which a viral vector infects tissues or cells other than the desired tissue or cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62 / 671,949, filed May 15, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] government support This invention was made with government support under RM1 HG008525 awarded by the National Institutes of Health and NHGRI CEGS CCV. The government has certain rights in this invention.

[0003] Technical field of the invention The present invention relates to viral vectors with altered tropism. [Background technology]

[0004] Background of the Invention Adeno-associated virus (AAV) is an attractive agent for use as a gene delivery vector.Its simple structure also makes it an attractive target for gene improvement programs.Nevertheless, there is a continuing need in the art to improve the delivery of DNA using AAV and other viral vectors.In particular, there is a need in the art for viral vectors with modified tissue or cell tropism. Summary of the Invention [Means for solving the problem]

[0005] Summary of the Invention Viral vectors with altered tropism are described herein. Such vectors provide an improvement in the degree of tissue targeting achievable with such vectors. In particular, viral vectors with capsid polypeptide mutations that alter the tropism of viral particles compared to particles with wild-type capsid polypeptides are described. Through systematic mutation of viral capsid polypeptides, specific amino acid residues and amino acid regions have been identified that, when mutated, increase or decrease the tropism of the virus for specific tissues or cell types. This provides the capsid polypeptide with the ability to increase the targeting of a specific tissue or cell type by the viral vector, or conversely, decrease the targeting of a specific tissue or cell type by the viral vector, depending on the site-specific mutation introduced. In fact, it has been found herein that incorporating amino acid changes that increase the tropism for a desired tissue can not only improve the tropism of the viral vector, but also improve nucleic acid delivery to a specific tissue or cell type by reducing the efficiency with which the viral vector infects tissues or cells other than the desired tissue or cell. Mutations that achieve both of these effects and their use to improve delivery to desired or target tissues or cell types are described herein. Furthermore, it has surprisingly been found that within a single viral capsid polypeptide, mutations that improve tropism for a desired or target tissue or cell type can be combined with mutations that decrease tropism for an undesired tissue or cell type, thereby further improving the targeting efficiency of the vector.

[0006] One aspect of the technology described herein provides a viral capsid polypeptide having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 1-7.

[0007] In one embodiment of any aspect, the tissue is heart, kidney, liver, lung, spleen, or blood.

[0008] In one embodiment of any aspect, the tropism for the tissue is increased. In one embodiment of any aspect, the tropism for the tissue is decreased.

[0009] Another aspect of the technology described herein provides a viral capsid polypeptide having a mutation corresponding to a mutation in the polypeptide of SEQ ID NO:1, wherein the mutation is selected from the group consisting of the mutations in Tables 1-9 relative to SEQ ID NO:1.

[0010] Yet another aspect of the technology described herein provides a viral capsid polypeptide, the viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO:2, wherein said region corresponding to the amino acid sequence of SEQ ID NO:2 comprises a mutation compared to SEQ ID NO:2 that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 10-15.

[0011] Another aspect of the technology described herein provides nucleic acids encoding any of the viral capsid polypeptides described herein.

[0012] Another aspect of the technology described herein provides a viral particle comprising any of the viral capsid polypeptides described herein.

[0013] Another aspect of the technology described herein provides a method of delivering nucleic acid to a cell, the method comprising contacting the cell with a viral particle comprising any of the viral capsid polypeptides described herein.

[0014] Another aspect of the technology described herein provides a method of delivering nucleic acid to a blood cell, the method comprising contacting the blood cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 1.

[0015] Another aspect of the technology described herein provides a method of delivering nucleic acid to a cardiac cell, the method comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2.

[0016] Another embodiment of the technology described herein provides a method of delivering nucleic acid to a kidney cell, the method comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3.

[0017] Another embodiment of the technology described herein provides a method of delivering nucleic acid to a liver cell, the method comprising contacting the liver cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 4.

[0018] Another embodiment of the technology described herein provides a method of delivering nucleic acid to a lung cell, the method comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5.

[0019] Another embodiment of the technology described herein provides a method of delivering nucleic acid to a spleen cell, the method comprising contacting the spleen cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 6.

[0020] In one embodiment of any aspect, delivery is at least 1.1 fold more efficient compared to the wild-type viral capsid polypeptide.

[0021] Another aspect of the technology described herein provides a method of reducing the tissue tropism of a virus comprising a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation as set forth in any of Tables 7-9 (Attenuation Tables).

[0022] Another aspect of the technology described herein provides a method of increasing delivery of nucleic acid to kidney, heart, or lung cells, comprising contacting kidney, heart, or lung cells with a viral particle comprising a viral capsid polypeptide that comprises a mutation that reduces delivery of nucleic acid to liver, blood, or spleen cells.

[0023] In one embodiment of any aspect, the mutation that reduces delivery of nucleic acids to cells of the liver, blood, or spleen is selected from any of Tables 7-9.

[0024] Another aspect of the technology described herein provides a method of delivering a nucleic acid to a lung cell, the method comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5 and a mutation selected from any of Tables 7-9.

[0025] Another aspect of the technology described herein provides a method of delivering a nucleic acid to a cardiac cell, the method comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2 and a mutation selected from any of Tables 7-9.

[0026] Another aspect of the technology described herein provides a method of delivering nucleic acid to a kidney cell, the method comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3 and a mutation selected from any of Tables 7-9.

[0027] Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in a region of amino acids selected from the group consisting of 440-460 of SEQ ID NO:1, 475-505 of SEQ ID NO:1, 518-532 of SEQ ID NO:1, and 560-590 of SEQ ID NO:1, which alters the tissue tropism of a virus comprising the viral capsid polypeptide.

[0028] Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in a region of amino acids 561 to 588 of SEQ ID NO: 1 that increases tissue tropism of the virus to the kidney, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIATTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 3); DEEEIRQTNPVATEGYGEVSTNLMHGNK (SEQ ID NO: 4); DEEEIRTTNPVATEQYGIVnStTNLNEGNR (SEQ ID NO: 5); DEEEIRTTNPVATECYGSVSTDLQSGNL (SEQ ID NO: 6); DENEIRTTNPVATEIYGSVSTeNLQNnGdNR (SEQ ID NO: 7); DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR (SEQ ID NO: 8); DEEEIRTTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 9).

[0029] Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases tissue tropism of the virus to the liver, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIRTTNPVATEQYGVVSDNLQRGNR (SEQ ID NO: 10); DECEIRTTNPVATEQYGSVGENLQRGNR (SEQ ID NO: 11); DEEEIRTTNPVATEQYGVVSENLQRGNR (SEQ ID NO: 12); DESEITTNPVATEQYGWVSTNQQRGNR (SEQ ID NO: 13); HELEIATTNPVATEQYGSASTNIQRGNR (SEQ ID NO: 14); DEEEIATTNPVATEQYGGVSTNLQRGNR (SEQ ID NO: 15).

[0030] Another aspect of the technology described herein provides an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases tissue tropism of the virus to the lung, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIVTTNPVATEQYGNVSTNLQRGNR (SEQ ID NO: 16); DEDEISTTNPVATEQYGSCSTNLQRGNR (SEQ ID NO: 17); QEEEIRTTNPVATEQYGSVSTNLQRGDR (SEQ ID NO: 18); NEEEIRTTNPCATEVYGSVSTNLQRGNR (SEQ ID NO: 19); DEQEIVTTNPVATEVYGTVSTNLQRGNR (SEQ ID NO: 20).

[0031] Another aspect of the technology described herein provides a method for altering the tropism of a virus comprising a capsid polypeptide corresponding to the polypeptide of SEQ ID NO:1, the method comprising introducing mutations into at least two regions selected from the group consisting of amino acids 440-460 of SEQ ID NO:1, amino acids 475-505 of SEQ ID NO:1, amino acids 518-532 of SEQ ID NO:1, and amino acids 560-590 of SEQ ID NO:1.

[0032] Another embodiment provides a viral capsid polypeptide having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the packaging efficiency of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in Table 16.

[0033] definition

[0034] As used herein, the term "altering tropism" refers to a mutation or set of mutations that alters the efficiency with which a viral vector delivers nucleic acid to a given tissue or cell type. The tropism of a virus or viral vector is generally determined by the structure of its outer surface that interacts with receptors or other cell surface determinants on target cells. For AAV vectors, in particular, the tropism of a viral vector is primarily determined by the viral capsid polypeptide, and as described herein, the tropism of such vectors can be altered by changing the amino acid sequence of the viral capsid polypeptide. An amino acid change that alters the efficiency of viral vector delivery of nucleic acid to a target cell or tissue type by at least 10% compared to a reference vector (often, but not necessarily, a wild-type vector) is an altered tropism. For clarity, an altered tropism can be an increase / enhancement of at least 10% (1.1x) or a decrease / weakness of at least 10% (0.9x).

[0035] As used herein, the term "increased tropism: or "increased tropism" refers to an increase in the efficiency of viral vector delivery of a nucleic acid to a target cell or tissue type by at least 0.1-fold or 10% compared to a reference vector. In various embodiments, the increase in the efficiency of viral vector delivery of a nucleic acid to a target cell or tissue is at least 1.1-fold (i.e., more than 10%), 1.2-fold (i.e., more than 20%), at least 1.5-fold (i.e., more than 50%), at least 2.0-fold (i.e., 2-fold), at least 5.0-fold, or at least 10.0-fold compared to a reference vector.

[0036] As used herein, the term "reduced tropism" or "reduced tropism" refers to a decrease in the efficiency of viral vector delivery of nucleic acid to target cells or tissue types by at least 10% compared to a reference vector. In various embodiments, the efficiency of viral vector delivery of nucleic acid to target cells or tissues is at most 0.9 times (10% or 90% lower than the reference), at most 0.8 times (20% or 80% lower than the reference), at most 0.7 times (30% or 70% lower than the reference), at most 0.5 times (50% or 50% lower than the reference), at most 0.3 times (70% or 30% lower than the reference), at most 0.2 times (80% or 20% lower than the reference), at most 0.1 times (90% or 10% lower than the reference) or lower compared to the reference vector.

[0037] As used herein, the term "corresponding," when used in reference to an amino acid or polynucleotide sequence, means that a given amino acid or polynucleotide sequence in one polypeptide or polynucleotide molecule has similar structural, functional, or both characteristics compared to an amino acid or polynucleotide sequence at an analogous position in another polypeptide or polynucleotide molecule. Homologs of a given polypeptide in different species "correspond" to each other, as do regions or domains of homologous polypeptides from different species. Similarly, capsid polypeptides of different serotypes of viral vectors, including but not limited to adeno-associated viral (AAV) vectors, "correspond" to each other, as do regions of such polypeptides defined, for example, by alignment of their amino acid sequences. Although other alignment parameters may be used to define such regions, for the avoidance of doubt, alignments may be performed using BLAST® (Basic Local Alignment Search Tool) using the default parameters of version BLAST+ 2.8.0, released March 28, 2018. The present invention provides the following aspects. (Item 1) A viral capsid polypeptide having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 1-9. (Item 2) 2. The viral capsid polypeptide of item 1, wherein the tissue is heart, kidney, liver, lung, spleen or blood. (Item 3) 2. The viral capsid polypeptide of item 1, wherein the tropism for the tissue is increased. (Item 4) 2. The viral capsid polypeptide of item 1, wherein the tropism for the tissue is reduced. (Item 5) A nucleic acid encoding the viral capsid polypeptide according to any one of items 1 to 4. (Item 6) A virus particle comprising the viral capsid polypeptide according to any one of items 1 to 4. (Item 7) A viral capsid polypeptide having a mutation corresponding to a mutation in the polypeptide of SEQ ID NO: 1, wherein the mutation is selected from the group consisting of the mutations in Tables 1-9 relative to SEQ ID NO: 1. (Item 8) 1. A viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO:2, wherein said region corresponding to the amino acid sequence of SEQ ID NO:2 comprises a mutation compared to SEQ ID NO:2 that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 10-15. (Item 9) 2. The viral capsid polypeptide of item 1, wherein the tissue is heart, kidney, liver, lung, spleen or blood. (Item 10) 9. The viral capsid polypeptide of item 8, wherein the tropism for the tissue is increased. (Item 11) 9. The viral capsid polypeptide of item 8, wherein the tropism for the tissue is reduced. (Item 12) A nucleic acid encoding the viral capsid polypeptide according to any one of items 8 to 11. (Item 13) A virus particle comprising the viral capsid polypeptide according to any one of items 8 to 11. (Item 14) 12. A method for delivering a nucleic acid to a cell, the method comprising contacting the cell with a viral particle comprising the viral capsid polypeptide according to items 1 to 4 or 8 to 11. (Item 15) A method of delivering nucleic acid to a blood cell, comprising contacting the blood cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 1. (Item 16) A method of delivering a nucleic acid to a cardiac cell, comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2. (Item 17) A method of delivering nucleic acid to a kidney cell, comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3. (Item 18) A method of delivering a nucleic acid to a liver cell, comprising contacting the liver cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 4. (Item 19) A method of delivering a nucleic acid to a lung cell, comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5. (Item 20) A method of delivering nucleic acid to a spleen cell, comprising contacting the spleen cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 6. (Item 21) 21. The method of any of items 14 to 20, wherein the delivery is at least 1.1 times more efficient compared to wild-type viral capsid polypeptide. (Item 22) A method for reducing the tissue tropism of a virus comprising a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation as set forth in any of Tables 7-9. (Item 23) A method for increasing delivery of nucleic acid to kidney, heart or lung cells, comprising contacting kidney, heart or lung cells with a viral particle comprising a viral capsid polypeptide containing a mutation that reduces delivery of nucleic acid to liver, blood or spleen cells. (Item 24) 24. The method of item 23, wherein the mutation that reduces delivery of nucleic acid to liver, blood, or spleen cells is selected from any of Tables 7 to 9. (Item 25) A method for delivering a nucleic acid to a lung cell, comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5 and a mutation selected from any of Tables 7-9. (Item 26) A method for delivering a nucleic acid to a cardiac cell, the method comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2 and a mutation selected from any of Tables 7-9. (Item 27) A method for delivering nucleic acid to a kidney cell, comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3 and a mutation selected from any of Tables 7-9. (Item 28) An AAV2 capsid polypeptide comprising a mutation in a region of amino acids selected from the group consisting of 440 to 460 of SEQ ID NO: 1, 475 to 505 of SEQ ID NO: 1, 518 to 532 of SEQ ID NO: 1, and 560 to 590 of SEQ ID NO: 1, which alters the tissue tropism of a virus comprising said viral capsid polypeptide. (Item 29) 29. The AAV2 capsid polypeptide of item 28, wherein the tissue is kidney, liver, or lung. (Item 30) 29. The AAV2 capsid polypeptide of item 28, wherein the tropism is increased. (Item 31) An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the kidney, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIATTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 3); DEEEIRQTNPVATEGYGEVSTNLMHGNK (SEQ ID NO: 4); DEEEIRTTNPVATEQYGIVnStTNLNEGNR (SEQ ID NO: 5); DEEEIRTTNPVATECYGSVSTDLQSGNL (SEQ ID NO: 6); DENEIRTTNPVATEIYGSVSTeNLQNnGdNR (SEQ ID NO: 7); DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR (SEQ ID NO: 8); DEEEIRTTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 9). (Item 32) An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the liver, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIRTTNPVATEQYGVVSDNLQRGNR (SEQ ID NO: 10); DECEIRTTNPVATEQYGSVGENLQRGNR (SEQ ID NO: 11); DEEEIRTTNPVATEQYGVVSENLQRGNR (SEQ ID NO: 12); DESEITTNPVATEQYGWVSTNQQRGNR (SEQ ID NO: 13); HELEIATTNPVATEQYGSASTNIQRGNR (SEQ ID NO: 14); DEEEIATTNPVATEQYGGVSTNLQRGNR (SEQ ID NO: 15). (Item 33) An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the lungs, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIVTTNPVATEQYGNVSTNLQRGNR (SEQ ID NO: 16); DEDEISTTNPVATEQYGSCSTNLQRGNR (SEQ ID NO: 17); QEEEIRTTNPVATEQYGSVSTNLQRGDR (SEQ ID NO: 18); NEEEIRTTNPCATEVYGSVSTNLQRGNR (SEQ ID NO: 19); DEQEIVTTNPVATEVYGTVSTNLQRGNR (SEQ ID NO: 20). (Item 34) 1. A method for altering the tropism of a virus comprising a capsid polypeptide corresponding to the polypeptide of SEQ ID NO: 1, the method comprising introducing mutations into at least two regions selected from the group consisting of amino acids 440 to 460 of SEQ ID NO: 1, amino acids 475 to 505 of SEQ ID NO: 1, amino acids 518 to 532 of SEQ ID NO: 1, and amino acids 560 to 590 of SEQ ID NO: 1. (Item 35) A viral capsid polypeptide having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the packaging efficiency of a virus containing said viral capsid polypeptide, wherein said mutation is selected from the mutations in Table 16. [Brief explanation of the drawings]

[0038] [Figure 1] Figure 1 shows a heatmap of all path numbers searched versus their distance from the wild type (e.g., number of mutations relative to the wild type). Beneficial, neutral, and deleterious mutations are shown. Most mutations are deleterious.

[0039] [Figure 2] Figure 2 shows a heatmap of some of the path numbers searched versus their distance from the wild type (e.g., number of mutations relative to the wild type). Beneficial, neutral, and deleterious mutations are shown. The heatmap highlights that longer paths result in earlier death.

[0040] [Figure 3] Figure 3 shows a chart plotting the fitness of the first mutation versus the distance at which fitness drops below wild type (e.g., when the virus is no longer viable). On the x-axis, 0.0 indicates a neutral mutation (e.g., a mutation that does not change the fitness of the virus), positive values ​​indicate beneficial mutations (e.g., a mutation that increases the fitness of the virus), and negative values ​​indicate deleterious mutations (e.g., a mutation that decreases the fitness of the virus). Values ​​on the y-axis indicate the number of mutations relative to the wild type.

[0041] [Figure 4]Figure 4 is a line graph of the pass rate showing at what mutation rate the mutation library fails. Random mutation results in a sharp decrease at one mutation compared to the wild type, whereas fast (e.g., deep search mutation) mutation can tolerate up to six mutations. Deep search mutation is over 40 times more efficient than random search at distances greater than or equal to six mutations compared to the wild type.

[0042] [Figure 5] FIG. 5 shows a heat map of amino acid insertions introduced into AAV2 viral capsid polypeptides that are beneficial, neutral, or deleterious to viral packaging.

[0043] [Figure 6] FIG. 6 shows a heat map of amino acid substitutions introduced into AAV2 viral capsid polypeptides that are beneficial, neutral, or deleterious to viral packaging. DETAILED DESCRIPTION OF THE INVENTION

[0044] Detailed Description of the Invention Viral vectors with altered tropism are described herein. Such vectors provide an improvement in the degree of tissue targeting achievable with such vectors. In particular, viral vectors with capsid polypeptide mutations that alter the tropism of viral particles compared to particles with wild-type capsid polypeptides are described. Through systematic mutation of viral capsid polypeptides, specific amino acid residues and amino acid regions have been identified that, when mutated, increase or decrease the tropism of the virus for specific tissues or cell types. This provides the capsid polypeptide with the ability to increase the targeting of a specific tissue or cell type by the viral vector, or conversely, decrease the targeting of a specific tissue or cell type by the viral vector, depending on the site-specific mutation introduced. In fact, it has been found herein that incorporating amino acid changes that increase the tropism for a desired tissue can not only improve the tropism of the viral vector, but also improve nucleic acid delivery to a specific tissue or cell type by reducing the efficiency with which the viral vector infects tissues or cells other than the desired tissue or cell. Mutations that achieve both of these effects and their use to improve delivery to desired or target tissues or cell types are described herein. Furthermore, it has surprisingly been found that within a single viral capsid polypeptide, mutations that improve tropism for a desired or target tissue or cell type can be combined with mutations that decrease tropism for an undesired tissue or cell type, thereby further improving the targeting efficiency of the vector.

[0045] Viruses typically have a tropism for a specific type of cell or tissue in the native host. This increased or decreased tropism can be determined by selection in the native host cells or tissues or by selection in non-native host cells or tissues. Indeed, experiments were performed to select for increased infectivity in the following tissues: blood, brain, heart, kidney, liver, lung, and spleen. Substitution, deletion, or insertion mutations that increased infectivity in these tissues were found. These determinations were also combined with the ability of mutant plasmids to be packaged into virions.

[0046] As detailed below, specific mutations were identified and evaluated for their effect on viral DNA packaging and viral infectivity in specific tissues. These specific mutations are in the capsid protein. Isolated and purified compositions containing mutant capsid proteins and the nucleic acids encoding them can be used for further viral improvement, viral preparation and manufacturing, and safety and efficacy studies. Once a mutant protein sequence is identified as beneficial, any one or more nucleic acid codons that specify such a protein sequence can be used. Mutations can be combined together in a single viral nucleic acid or single viral protein sequence for improved properties. Below are described mutations in viral capsid polypeptides that enable viral genome packaging but, when combined, positively, negatively, or both alter the tropism of the viral vector relative to a given tissue or cell type. Methods for using mutant viral capsid polypeptides and viral vectors containing them to introduce nucleic acids into desired tissues or cell types with improved selectivity for those tissues or cell types are also described. Below are provided descriptions of various mutations and considerations for their use to generate viral vectors with improved properties. Generation of mutants and virus particles

[0047] A DNA library of AAV capsid mutants was created. Initially, each mutant capsid with a single mutation was generated. All possible single amino acid substitutions, insertions, and deletions of AAV2 were generated. In subsequent steps, several mutations were combined within the capsid gene. The library of AAV2 capsid gene sequence variants was cloned into a plasmid containing the AAV inverted terminal repeat (ITR). The final ITR plasmid contained a cytomegalovirus (CMV) promoter upstream of the Cap gene.

[0048] An AAV viral library was generated from the DNA library. The capsid library plasmid, AAV pHelper plasmid, and a plasmid containing the AAV2 Rep gene were co-transfected into HEK-293 cells using PEI. Capsids were purified using standard cell lysis techniques (freeze-thaw or addition of 5M NaCl), benzonase treatment to remove unpackaged genomes, and purification and concentration by iodixanol ultracentrifugation.

[0049] The in vivo packaging ability of viral capsid mutants was measured. The number of packaged viruses ("viruses") relative to the number of input viral genomes ("plasmids") was determined. Measuring the frequency of capsid (or other library component) mutants before and after selection reveals which mutations are beneficial and which are deleterious, based on the particular selection method. Assessment of viral tropism

[0050] To investigate viral tropism, the viral library was injected intravenously into mice. Blood was collected 1 hour after injection, and tissue samples were collected 1–2 weeks after injection. Viral DNA was extracted from bulk biological samples using standard techniques.

[0051] The frequencies of capsid variants in biological samples, viral libraries, and DNA libraries were measured using high-throughput DNA sequencing. Mutant frequencies were normalized by dividing the number of reads matching each variant by the number of reads matching the AAV2 WT sequence in each sample.

[0052] A selection value indicating enhancement or attenuation in a particular sample is calculated relative to the initial library. A selection value greater than 1 indicates enhancement relative to WT. A selection value less than 1 indicates attenuation relative to WT.

[0053] For packaging selection, the frequency in the viral library was compared with that in the DNA library as a reference. Enrichment means that the mutant was packaged more efficiently than the WT.

[0054] For tissue selection, the frequency in the tissue sample was compared with that in the viral library as a reference. Enrichment means that the mutant was delivered to a specific tissue more efficiently than the WT. Attenuation means that the mutant was delivered to a specific tissue less efficiently than the WT. Viral Capsid Polypeptides

[0055] Viral tropism refers to the cell or tissue types in a host that recruit a virus and support its growth. Various factors affect viral tropism, including the expression of cell surface receptors and / or ligands, the expression of transcription factors, and the expression of tropogens (e.g., cell surface glycoproteins). One aspect of the present invention provides a viral capsid polypeptide having a mutation compared to wild-type adeno-associated virus 2 (AAV2, e.g., SEQ ID NO: 1) that alters the tissue tropism of a virus comprising the viral capsid polypeptide, wherein the mutation is selected from the mutations in any one of Tables 1-9. In one embodiment, the tissue is blood, heart, kidney, liver, lung, or spleen.

[0056] In one embodiment, the tropism is increased, e.g., a virus comprising a mutant viral capsid polypeptide delivers nucleic acid to a target cell type more efficiently than a virus comprising a wild-type viral capsid polypeptide. In one embodiment, the tropism is at least 1.1-fold more efficient (e.g., 10% more efficient than the reference level or 110% more efficient than the reference level) than a wild-type viral capsid polypeptide. In one embodiment, nucleic acid delivery is at least 1.5-fold, at least 2-fold, at least 4-fold, at least 5-fold, at least 10-fold, or more efficient than a virus comprising a wild-type viral capsid polypeptide. One of ordinary skill in the art can measure the delivery efficiency of a viral particle comprising any of the viral capsid polypeptides described herein, for example, using a PCR bead assay on an isolated target cell or tissue type (e.g., blood, heart, kidney, liver, lung, or spleen) to assess whether the nucleic acid is expressed in the target cell. The expression of nucleic acid delivered by a viral particle comprising any of the viral capsid polypeptides described herein or a wild-type viral capsid polyprotein can be compared to determine the change in expression as a measure of delivery efficiency.

[0057] In one embodiment, the tropism is reduced, e.g., a virus comprising a mutant viral capsid polypeptide delivers nucleic acid to a target cell type less efficiently than a virus comprising a wild-type viral capsid polypeptide. In one embodiment, the tropism is reduced by at least 10%. In other embodiments, the tropism is reduced by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or more compared to a virus comprising a wild-type viral capsid polypeptide. Methods for measuring tropism are described herein above.

[0058] The following describes single amino acid capsid changes that increase the tropism of a virus (e.g., AAV2) for a given tissue or cell type, such as blood, heart, kidney, liver, lung, or spleen. In one embodiment, a single mutation described herein is introduced into the amino acid sequence of the wild-type AAV2 capsid protein (e.g., SEQ ID NO: 1) to increase or decrease tissue or cell type tropism or virus.

[0059] SEQ ID NO: 1 is the amino acid sequence encoding the wild-type AAV2 capsid protein. [ka]

[0060] As used herein, "mutation" refers to any change in an amino acid sequence, such as the substitution, insertion, or deletion of at least one amino acid. For the mutations described herein, a substitution at a particular position is indicated by the wild-type amino acid, followed by the position of the substituted amino acid, followed by the identity of the substituted amino acid in parentheses; for example, Q101(A) means that glutamine is substituted with alanine at position 101. When multiple amino acids can be substituted at a particular position, the parentheses list all possible single substitutions; for example, P31(IKRT) means that proline at position 31 is substituted with any one of isoleucine, lysine, arginine, or threonine. To indicate an insertion, the amino acid position contains a decimal number followed by the amino acid; the amino acid following the decimal number is inserted after the indicated position; for example, 28.5(AGVY) means that any one of alanine, glycine, valine, or tyrosine is inserted immediately after amino acid 28 and immediately before amino acid 29. Amino acid deletions: A "(-)" follows the amino acid position, for example, A35(-) means that amino acid 35 (alanine) is deleted from the sequence.

[0061] The following provide mutations to the AAV2 capsid polypeptide of SEQ ID NO:1 that confer altered tropism to the indicated degree for cells of the indicated tissues. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2] [Table 3-1] [Table 3-2] [Table 4] [Table 5-1] [Table 5-2] [Table 6] [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4] [Table 7-5] [Table 7-6]

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

Table 7-20

Table 7-21

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

[0062] The above describes a wide range of individual amino acid mutations that are tolerated and have a positive or negative effect on viral tropism. It is specifically contemplated herein that these various mutations can be combined to further alter viral tropism or other characteristics. In one embodiment, for example, at least two of the single amino acid mutations described herein are introduced in combination. For example, two mutations that affect tropism to the liver can be combined. The resulting virus is also expected to have increased tropism to the liver. In a further embodiment, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more of the mutations described herein are introduced in combination. The inventors have demonstrated herein that a wide range of mutations is tolerated, however, no more than 40% of the amino acids should be changed compared to the reference sequence (e.g., SEQ ID NO: 1). In certain embodiments, 35% or less, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the amino acids are changed compared to a reference sequence (e.g., SEQ ID NO: 1).

[0063] Mutations at equivalent positions in other homologous viruses can be created and used to improve viral packaging and infectivity. Examples of other homologous viruses include AAV serotypes 1 and 3-12, as well as other natural isolates or synthetic sequences. Corresponding positions in homologous viruses can be inferred from sequence homology with AAV2. In one embodiment, the mutations described herein are introduced into the corresponding amino acid sequences of AAV1, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, and AAV13 capsid polypeptides.

[0064] Site-directed mutagenesis is known in the art and can be used to introduce point mutations (e.g., amino acid substitutions, insertions, or deletions) or other mutations or combinations thereof into viral capsid polypeptides. Site-directed mutagenesis is described, for example, in Li B et al., Hum Gene Ther Methods. 2015 Dec;26(6):211-20 and Bachman, J. Methods Enzymol. 2013;529:241-248, which are incorporated herein by reference in their entireties.

[0065] The combination of mutations can be used to achieve specific vector targeting goals.For example, it may be desirable to use a virus that has improved nucleic acid delivery in a first tissue and reduced nucleic acid delivery in one or more second tissues.This virus can have increased tissue specificity, whether achieved by single mutation or multiple mutations.

[0066] Mutable subregions of the AAV2 capsid protein

[0067] It has been found herein that certain regions of the AAV2 capsid protein tolerate amino acid changes, such as deletions, insertions, or substitutions, better than others, and that changes to certain regions that tolerate such changes have a pronounced effect on viral vector tropism. As shown in the tables herein, the region of the AAV2 capsid polypeptide of SEQ ID NO: 1, amino acids 440-600, is tolerant of a variety of changes and is highly influential on viral tropism, with specific mutations identified herein as affecting tropism toward or away from specific tissues or cell types. Within this region, subregions including amino acids 440-460, 475-505, 518-532, and 560-590 have been found to be particularly important for altering viral tropism, for example, in the heatmaps provided herein, and one of these subregions, amino acids 561-588, has been shown herein to tolerate a wide range of mutations. This region, found in single amino acid mutation studies to be important for tropism, was selected for initial studies of the effects of combinations of mutations. As shown in SEQ ID NOS: 3-20, combinations of two to at least eight different single amino acid mutations within this region were found to be well tolerated and further affected tropism.

[0068] SEQ ID NO:2 is an amino acid sequence encoding the amino acid region from amino acids 440 to 600 of SEQ ID NO:1. [ka] [Table 10-1] [Table 10-2] [Table 10-3] [Table 11] [Table 12-1] [Table 12-2] [Table 13] [Table 14] [Table 15]

[0069] Interestingly, when mutation combinations were examined, it was found that when a single mutation identified as beneficial, for example in altering tropism, was introduced, the resulting polypeptide tended to tolerate further mutations to include other single amino acid changes that were also found to be beneficial. This is illustrated, for example, in Figure 3, which plots the fitness of the first mutation versus the number of mutations away from the wild type that the virus can tolerate before it is no longer viable. On the x-axis is the fitness of the first mutation (i.e., whether the single mutation is neutral, deleterious, or beneficial). On the y-axis is the number of mutations that the virus can tolerate before it is no longer viable. This figure shows that when a virus has a beneficial first mutation, it tolerates significantly more mutations than viruses with neutral or deleterious first mutations. Therefore, combinations of mutations that do not interfere with viral fitness are expected to be well tolerated.

[0070] Subregions within viral capsid polypeptides (e.g., regions within full-length polypeptides) that are likely to tolerate amino acid changes have been further identified herein. One aspect of the present specification is an AAV2 capsid polypeptide comprising a mutation in a subregion of amino acids selected from the group consisting of 440-460 of SEQ ID NO:1, 475-505 of SEQ ID NO:1, 518-532 of SEQ ID NO:1, and 560-590 of SEQ ID NO:1, which alters the tissue tropism of a virus comprising the viral capsid polypeptide. In further embodiments, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more of the mutations described herein are introduced in combination into a subregion. While a wide range of mutations is tolerated, no more than 40% of the amino acids within a subregion should be altered compared to a reference sequence (e.g., SEQ ID NO:1). In certain embodiments, less than 35%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the amino acids within a subregion are altered compared to a reference sequence (e.g., SEQ ID NO: 1).

[0071] In one embodiment, the mutations described herein increase viral tropism more efficiently when introduced into a specific subregion of a viral capsid polypeptide. In one embodiment, delivery is at least 1.1 times more efficient than that of a wild-type viral capsid polypeptide. In another embodiment, delivery of nucleic acid is at least 1.5 times, at least 2 times, at least 4 times, at least 5 times, at least 10 times, or more efficient than that of a virus comprising a wild-type SEQ ID NO: 1 capsid polypeptide.

[0072] One aspect of the technology is a method for altering the tropism of a virus, the method comprising modifying a capsid polypeptide corresponding to the polypeptide of SEQ ID NO: 1 to introduce mutations into at least two regions selected from the group consisting of amino acids 440 to 460 of SEQ ID NO: 1, amino acids 475 to 505 of SEQ ID NO: 1, amino acids 518 to 532 of SEQ ID NO: 1, and amino acids 560 to 590 of SEQ ID NO: 1.

[0073] One aspect is an AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the kidney, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIATTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 3); DEEEIRQTNPVATEGYGEVSTNLMHGNK (SEQ ID NO: 4); DEEEIRTTNPVATEQYGIVnStTNLNEGNR (SEQ ID NO: 5); DEEEIRTTNPVATECYGSVSTDLQSGNL (SEQ ID NO: 6); DENEIRTTNPVATEIYGSVSTeNLQNnGdNR (SEQ ID NO: 7); DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR (SEQ ID NO: 8); DEEEIRTTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 9).

[0074] An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the liver, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIRTTNPVATEQYGVVSDNLQRGNR (SEQ ID NO: 10); DECEIRTTNPVATEQYGSVGENLQRGNR (SEQ ID NO: 11); DEEEIRTTNPVATEQYGVVSENLQRGNR (SEQ ID NO: 12); DESEITTNPVATEQYGWVSTNQQRGNR (SEQ ID NO: 13); HELEIATTNPVATEQYGSASTNIQRGNR (SEQ ID NO: 14); DEEEIATTNPVATEQYGGVSTNLQRGNR (SEQ ID NO: 15).

[0075] An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the lungs, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIVTTNPVATEQYGNVSTNLQRGNR (SEQ ID NO: 16); DEDEISTTNPVATEQYGSCSTNLQRGNR (SEQ ID NO: 17); QEEEIRTTNPVATEQYGSVSTNLQRGDR (SEQ ID NO: 18); NEEEIRTTNPCATEVYGSVSTNLQRGNR (SEQ ID NO: 19); DEQEIVTTNPVATEVYGTVSTNLQRGNR (SEQ ID NO: 20).

[0076] One aspect of the technology described herein is a nucleic acid encoding any of the viral capsid polypeptides described herein.

[0077] Another aspect of the technology described herein is a viral particle comprising any of the viral capsid polypeptides described herein. Corresponding mutations

[0078] For example, mutations at corresponding positions in homologous viral capsid polypeptides are expected to have similar effects on viral tropism. Corresponding positions can include, for example, positions relative to a full-length capsid polypeptide (e.g., SEQ ID NO: 1), or even positions relative to a subdomain or subregion of a full-length capsid polypeptide (e.g., SEQ ID NO: 2). In one aspect, provided herein is a viral capsid polypeptide having a mutation corresponding to a mutation in the polypeptide of SEQ ID NO: 1 described herein, or a mutation selected from the group consisting of a mutation in Tables 1-9 relative to SEQ ID NO: 1. In one embodiment, the homologous viral capsid polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to the wild-type AAV2 capsid protein (e.g., SEQ ID NO: 1). In another embodiment, viral capid polypeptides of AAV serotypes 1 and 3-13 are homologous to the AAV2 capsid polypeptide.

[0079] One aspect described herein is a viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO:2, wherein said region corresponding to the amino acid sequence of SEQ ID NO:2 comprises a mutation compared to SEQ ID NO:2 that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 10-15. In one embodiment, the homologous viral capsid polypeptide has at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO:2.

[0080] As described elsewhere herein, identification of amino acid sites or regions of the amino acid sequence in the AAV2 capsid polypeptide of SEQ ID NO: 1 that are generally tolerant of change (i.e., that allow packaging and infection by viral particles), as well as regions that positively or negatively affect tropism and / or packaging, can be used to induce changes in capsid polypeptides of other AAV serotypes to similarly affect function or provide modified properties. Thus, through the use of sequence alignment, a mutation or set of mutations that provides a desired change in tropism and / or packaging of the AAV2 capsid polypeptide of SEQ ID NO: 1 can be introduced into corresponding positions in the capsid polypeptide of another AAV serotype to similarly affect those properties in that serotype. By way of example only, it has been determined herein that the region from amino acids 440 to 600 of the AAV2 capsid polypeptide of SEQ ID NO: 1 is important in determining viral tropism, and indeed, there are subregions within that region, e.g., amino acids 561 to 588, that strongly influence tropism. Introduction of the changes identified herein in this amino acid 440-600 region or amino acid 561-588 subregion of the AAV2 capsid polypeptide into the corresponding region or subregion of another AAV serotype capsid polypeptide will affect the tropism of that AAV serotype in a similar manner. Virus packaging

[0081] One embodiment of the technology described herein relates to a method for increasing viral packaging by modifying an AAV2 capsid polypeptide to include at least one mutation in a region of amino acids selected from the group consisting of 34-38 of SEQ ID NO:1, 133-152 of SEQ ID NO:1, 188-194 of SEQ ID NO:1, and 654-662 of SEQ ID NO:1.

[0082] One embodiment of the technology described herein relates to a method for increasing viral packaging efficiency by modifying an AAV2 capsid polypeptide to include any non-positive amino acid insertion, substitution, or deletion in a region of amino acids selected from the group consisting of 442-452 of SEQ ID NO: 1, 489-506 of SEQ ID NO: 1, 541-551 of SEQ ID NO: 1, and 577-596 of SEQ ID NO: 1. In another embodiment, packaging efficiency is increased by mutating multiple amino acids in a region of amino acids selected from the group consisting of 442-452 of SEQ ID NO: 1, 489-506 of SEQ ID NO: 1, 541-551 of SEQ ID NO: 1, and 577-596 of SEQ ID NO: 1 to any non-positive amino acid insertion, substitution, or deletion. [Table 16-1] [Table 16-2] [Table 16-3] [Table 16-4]

[0083] In one embodiment, a mutation in a viral capsid polypeptide that alters viral tropism described herein is combined with one or more mutations that increase viral packaging (e.g., any of the mutations in Table 16).

[0084] In another embodiment, a mutation in a viral capsid polypeptide that alters viral tropism described herein is combined with a mutation in a region of amino acids selected from the group consisting of 34-38 of SEQ ID NO:1, 133-152 of SEQ ID NO:1, 188-194 of SEQ ID NO:1, and 654-662 of SEQ ID NO:1 that increases viral packaging.

[0085] In yet another embodiment, a mutation in a viral capsid polypeptide that alters viral tropism described herein is combined with any non-positive amino acid insertion, substitution, or deletion in the region of amino acids selected from the group consisting of 442-452 of SEQ ID NO:1, 489-506 of SEQ ID NO:1, 541-551 of SEQ ID NO:1, and 577-596 of SEQ ID NO:1 that increases viral packaging.

[0086] Any region in the packaging heatmap is tolerant of variation; for example, a neutral or positive region could potentially tolerate the insertion of additional functional peptide sequences, such as epitopes, tags, ligands, or other structural sequences that do not necessarily disrupt viral packaging. Methods for delivering nucleic acids

[0087] Provided herein are methods for delivering nucleic acids to cells, comprising contacting the cells with viral particles comprising any of the viral capsid polypeptides described herein. In one embodiment, the contacting occurs in vitro. In one embodiment, the contacting occurs ex vivo. In another embodiment, the contacting occurs in vivo, e.g., via local or systemic administration.

[0088] In one embodiment, delivery of the nucleic acid is at least 1.1 times or more efficient compared to the wild-type viral capsid polypeptide. In one embodiment, delivery of the nucleic acid is at least 1.5 times, at least 2 times, at least 4 times, at least 5 times, at least 10 times or more efficient compared to the wild-type viral capsid polypeptide. Methods for measuring tropism are described herein above.

[0089] In one embodiment, the nucleic acid is delivered to a blood cell by contacting the blood cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 1. The cell can be any type of blood cell. Exemplary blood cells include, but are not limited to, red blood cells, platelets, neutrophils, eosinophils, basophils, lymphocytes, or monocytes.

[0090] In one embodiment, the nucleic acid is delivered to a cardiac cell by contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2. Exemplary cardiac cells include, but are not limited to, cardiomyocytes, endocardial cells, or cardiac smooth muscle cells.

[0091] In one embodiment, the nucleic acid is delivered to a kidney cell by contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3. Exemplary kidney cells include, but are not limited to, kidney glomerular parietal cells, kidney glomerular podocytes, kidney proximal tubule brush border cells, Henle's loop slice cells, thick ascending limb cells, kidney distal tubule cells, collecting duct principal cells, collecting duct interstitial cells, and interstitial kidney cells.

[0092] In one embodiment, the nucleic acid is delivered to a liver cell by contacting the liver cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 4. Exemplary liver cells include, but are not limited to, parenchymal cells, non-parenchymal cells, sinusoidal endothelial cells, phagocytic Kupffer cells, hepatic stellate cells, and intrahepatic lymphocytes.

[0093] In one embodiment, the nucleic acid is delivered to a lung cell by contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5. Exemplary lung cells include, but are not limited to, bronchial, pulmonary epithelial, pulmonary smooth muscle, and alveolar cells.

[0094] In one embodiment, the nucleic acid is delivered to a spleen cell by contacting the spleen cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 6. Exemplary spleen cells include, but are not limited to, spleen endothelial cells and spleen fibroblasts. Methods for reducing viral tropism

[0095] One aspect of the technology described herein is a method for reducing the tissue tropism of a virus, the method comprising modifying a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1 by introducing a mutation set forth in any of Tables 7-9. In one embodiment, the viral tropism for the liver, spleen, or blood is reduced.

[0096] In one embodiment, the method for reducing viral tropism further comprises any mutation or combination thereof described herein that increases the tropism of tissues other than the tissue that reduces tropism.For example, the viral capsid polypeptide that has the mutation that reduces the tropism of the liver is combined with the mutation that increases the tropism of the kidney.In this example, it is intended that this combination of mutations will result in an increase in the tropism of the kidney.

[0097] Furthermore, one aspect provides a method for increasing delivery of nucleic acid to kidney, heart, or lung cells, comprising contacting kidney, heart, or lung cells with a viral particle comprising a viral capsid polypeptide comprising a mutation that reduces delivery of nucleic acid to liver, blood, or spleen cells. In one embodiment, the mutation that reduces delivery of nucleic acid to liver, blood, or spleen cells is selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

[0098] Another aspect provides a method of delivering nucleic acid to a lung cell, the method comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

[0099] Another aspect provides a method of delivering nucleic acid to a cardiac cell, the method comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

[0100] Yet another aspect provides a method of delivering nucleic acid to a kidney cell, the method comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3 and a mutation selected from any of Tables 7-9. In one embodiment, the contacting is performed in vivo, e.g., via local or systemic administration.

[0101] The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples, which are provided herein for illustrative purposes only and are not intended to limit the scope of the present invention.

[0102] Some embodiments of the technology described herein may be defined according to any of the following numbered paragraphs: 1. A viral capsid polypeptide having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 1-9. 2. The viral capsid polypeptide of paragraph 1, wherein the tissue is heart, kidney, liver, lung, spleen, or blood. 3. The viral capsid polypeptide of any of the preceding paragraphs, wherein said tropism for said tissue is increased. 4. The viral capsid polypeptide of any of the preceding paragraphs, wherein said tropism for said tissue is reduced. 5. A nucleic acid encoding a viral capsid polypeptide according to any of the preceding paragraphs. 6. A viral particle comprising a viral capsid polypeptide according to any of the preceding paragraphs. 7. A viral capsid polypeptide having a mutation corresponding to a mutation in the polypeptide of SEQ ID NO:1, wherein the mutation is selected from the group consisting of the mutations in Tables 1-9 relative to SEQ ID NO:1. 8. A viral capsid polypeptide comprising a region corresponding to the amino acid sequence of SEQ ID NO:2, wherein said region corresponding to the amino acid sequence of SEQ ID NO:2 comprises a mutation compared to SEQ ID NO:2 that alters the tissue tropism of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in any one of Tables 10-15. 9. The viral capsid polypeptide of any of the preceding paragraphs, wherein the tissue is heart, kidney, liver, lung, spleen, or blood. 10. The viral capsid polypeptide of any of the preceding paragraphs, wherein said tropism for said tissue is increased. 11. The viral capsid polypeptide of any of the preceding paragraphs, wherein said tropism for said tissue is reduced. 12. A nucleic acid encoding a viral capsid polypeptide according to any of the preceding paragraphs. 13. A viral particle comprising a viral capsid polypeptide according to any of the preceding paragraphs. 14. A method for delivering nucleic acid to a cell, comprising contacting said cell with a viral particle comprising a viral capsid polypeptide according to any of the preceding paragraphs. 15. A method for delivering nucleic acid to a blood cell, comprising contacting the blood cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 1. 16. A method for delivering nucleic acid to a cardiac cell, comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2. 17. A method for delivering nucleic acid to a kidney cell, comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3. 18. A method for delivering nucleic acid to a liver cell, comprising contacting the liver cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 4. 19. A method for delivering nucleic acid to a lung cell, comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5. 20. A method of delivering nucleic acid to a spleen cell, comprising contacting the spleen cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 6. 21. The method of any of the preceding paragraphs, wherein the delivery is at least 1.1-fold more efficient compared to wild-type viral capsid polypeptide. 22. A method for reducing the tissue tropism of a virus comprising a viral capsid polypeptide corresponding to the viral capsid polypeptide of SEQ ID NO: 1, the method comprising introducing a mutation as set forth in any of Tables 7-9. 23. A method for increasing delivery of nucleic acid to kidney, heart or lung cells, comprising contacting kidney, heart or lung cells with a viral particle comprising a viral capsid polypeptide that contains a mutation that reduces delivery of nucleic acid to liver, blood or spleen cells. 24. The method of paragraph 23, wherein the mutation that reduces delivery of nucleic acid to cells of the liver, blood, or spleen is selected from any of Tables 7-9. 25. A method for delivering nucleic acid to a lung cell, comprising contacting the lung cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 5 and a mutation selected from any of Tables 7-9. 26. A method for delivering nucleic acid to a cardiac cell, comprising contacting the cardiac cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 2 and a mutation selected from any of Tables 7-9. 27. A method for delivering nucleic acid to a kidney cell, comprising contacting the kidney cell with a viral particle comprising a viral capsid polypeptide comprising a mutation in Table 3 and a mutation selected from any of Tables 7-9. 28. An AAV2 capsid polypeptide comprising a mutation in a region of amino acids selected from the group consisting of 440-460 of SEQ ID NO:1, 475-505 of SEQ ID NO:1, 518-532 of SEQ ID NO:1 and 560-590 of SEQ ID NO:1, which alters the tissue tropism of a virus comprising said viral capsid polypeptide. 29. The AAV2 capsid polypeptide of paragraph 28, wherein the tissue is kidney, liver, or lung. 30. The AAV2 capsid polypeptide of paragraph 28, wherein the tropism is increased. 31. An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the kidney, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIATTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 3); DEEEIRQTNPVATEGYGEVSTNLMHGNK (SEQ ID NO: 4); DEEEIRTTNPVATEQYGIVnStTNLNEGNR (SEQ ID NO: 5); DEEEIRTTNPVATECYGSVSTDLQSGNL (SEQ ID NO: 6); DENEIRTTNPVATEIYGSVSTeNLQNnGdNR (SEQ ID NO: 7); DEEEIRTTNPVATEQYGSVSeTNpLvQNGdDR (SEQ ID NO: 8); DEEEIRTTNPVATEQYGDVSENLMHFQN (SEQ ID NO: 9). 32. An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the liver, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIRTTNPVATEQYGVVSDNLQRGNR (SEQ ID NO: 10); DECEIRTTNPVATEQYGSVGENLQRGNR (SEQ ID NO: 11); DEEEIRTTNPVATEQYGVVSENLQRGNR (SEQ ID NO: 12); DESEITTNPVATEQYGWVSTNQQRGNR (SEQ ID NO: 13); HELEIATTNPVATEQYGSASTNIQRGNR (SEQ ID NO: 14); DEEEIATTNPVATEQYGGVSTNLQRGNR (SEQ ID NO: 15). 33. An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561 to 588 of SEQ ID NO: 1 that increases the tissue tropism of the virus to the lung, wherein the mutated region of amino acids 561 to 588 of SEQ ID NO: 1 has a sequence selected from the group consisting of DEEEIVTTNPVATEQYGNVSTNLQRGNR (SEQ ID NO: 16); DEDEISTTNPVATEQYGSCSTNLQRGNR (SEQ ID NO: 17); QEEEIRTTNPVATEQYGSVSTNLQRGDR (SEQ ID NO: 18); NEEEIRTTNPCATEVYGSVSTNLQRGNR (SEQ ID NO: 19); DEQEIVTTNPVATEVYGTVSTNLQRGNR (SEQ ID NO: 20). 34. A method for altering the tropism of a virus comprising a capsid polypeptide corresponding to the polypeptide of SEQ ID NO:1, the method comprising introducing mutations into at least two regions selected from the group consisting of amino acids 440 to 460 of SEQ ID NO:1, amino acids 475 to 505 of SEQ ID NO:1, amino acids 518 to 532 of SEQ ID NO:1, and amino acids 560 to 590 of SEQ ID NO:1. 35. A viral capsid polypeptide, having a mutation compared to SEQ ID NO:1 (WT AAV2) that alters the packaging efficiency of a virus comprising said viral capsid polypeptide, wherein said mutation is selected from the mutations in Table 16.

[0103] References 1.Adachi,Kei,Enoki,Tatsuji,Kawano,Yasuhiro, Veraz,Michael,and Nakai Hiroyuki,Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.Nature Communications 5,3075(2014) 2. Grimm, D. and Zolotukhin, SE Pluribus Unum: 50 Years of Research, Millions of Viruses, and One Goal-Tailored Acceleration of AAV Evolution. Molecular Therapy 23, 1819-1831 (2015).

Claims

1. 1. An AAV2 capsid polypeptide, comprising:

1. An AAV2 capsid polypeptide comprising a mutation in the region of amino acids 561-588 of SEQ ID NO:1 that increases the tissue tropism of the virus to the kidney, wherein the region of amino acids 561-588 of SEQ ID NO:1 is replaced with a sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:

9.

2. A nucleic acid encoding the AAV2 capsid polypeptide of claim 1.

3. A viral particle comprising the AAV2 capsid polypeptide of claim 1.

4. 10. An in vitro method for delivering heterologous nucleic acid to a cell, the method comprising contacting the cell with a viral particle comprising the heterologous nucleic acid and an AAV2 capsid polypeptide of claim 1.

5. The in vitro method of claim 4 , wherein the cells are kidney cells.

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