NOVEL AAV LIBRARY

Abstract

An AAV library, comprising AAV variants having an amino acid sequence corresponding to the position amino acids 585 to 597 or 598 of AAV8 or the position amino acids 583 to 595 or 596 of AAV9, and the polynucleotide, host cells, thereof. A method of generating and screening an AAV library and its use.

Claims

1. An AAV library comprising a multitude of AAV variants, wherein each AAV variant comprise a variant of native AAV8 or AAV9 capsid protein comprising a substituted amino acid sequence relative to native AAV 8 or AAV9 capsid protein, the substituted amino acid sequence is located at VR VIII region of the native AAV 8 or AAV9 capsid protein, the native AAV 8 is with an amino acid sequence of SEQ ID NO:1, the native AAV 9 is with an amino acid sequence of SEQ ID NO:43.

2. The AAV library of claim 1, wherein the substituted amino acid sequence is located at amino acid position 585 to 597 or 585 to 598 of SEQ ID NO:1; or at the amino acids corresponding to amino acid position 583 to 595 or 583 to 596 of SEQ ID NO:43.

3. (canceled)

4. The library of claim 2, wherein the substituted sequence located the position amino acids 585 to 598 of SEQ ID NO:1 is:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14, wherein  Formula I: X.sub.1 is Asn, or Tyr, X.sub.2 is Leu, or Asn, or Gln, or Lys, or His, or Phe, X.sub.3 is Gln, or Asn, X.sub.4 is Gln, or Asn, or Ser, or Ala, or Asp, or Gly, X.sub.5 is Gln, or Thr, or Ala, or Gly, or Ser, or Asn, X.sub.6 is Asn, or Ala, or Ser, or Asp, or Thr, or Gln, X.sub.7 is Thr, or Ser, or Ala, or Arg, or Glu, or Gly, X.sub.8 is Ala, or Gln, or Asp, or Gly, or Arg, or Thr, X.sub.9 is Pro, or Ala, or Thr, X.sub.10 is Gln, or Thr, or Ala, or Ile, or Ser, or Asp, X.sub.11 is Ile, or Ala, or Thr, or Val, or Thr, or Ser, or Tyr X.sub.12 is Gly, or Gln, or Ser, or Ala, or Glu, X.sub.13 is Thr, or Ala, or Leu, or Asp, or Ser, or Asn, or Val, or Trp, or Met, X.sub.14 is Val, or Asp, the sequence doesn't comprise an amino acids sequence of SEQ ID NO:2.

5. The library of claim 4, wherein the substituted sequence is selected from SEQ ID NO: 3-42.

6. (canceled)

7. The AAV library of claim 2, wherein the substituted sequence located at the position amino acids 583 to 596 of SEQ ID NO:43 is:
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.sub.11X.sub.12X.sub.13X.sub.14, wherein  Formula I: X.sub.1 is Asn, or Tyr, X.sub.2 is Leu, or Asn, or Gln, or Lys, or His, or Phe, X.sub.3 is Gln, or Asn, X.sub.4 is Gln, or Asn, or Ser, or Ala, or Asp, or Gly, X.sub.5 is Gln, or Thr, or Ala, or Gly, or Ser, or Asn, X.sub.6 is Asn, or Ala, or Ser, or Asp, or Thr, or Gln, X.sub.7 is Thr, or Ser, or Ala, or Arg, or Glu, or Gly, X.sub.8 is Ala, or Gln, or Asp, or Gly, or Arg, or Thr, X.sub.9 is Pro, or Ala, or Thr, X.sub.10 is Gln, or Thr, or Ala, or Ile, or Ser, X.sub.11 is Ile, or Ala, or Thr, or Val, or Thr, or Ser, or Tyr X.sub.12 is Gly, or Gln, or Ser, or Ala, or Glu, X.sub.13 is Thr, or Ala, or Leu, or Asp, or Ser, or Asn, or Val, or Trp, or Met, X.sub.14 is Val, or Asp, the sequence doesn't comprise an amino acids sequence of SEQ ID NO:33.

8. The library of claim 7, wherein the substituted sequence is selected from SEQ ID NO: 3-42.

9. A library of polynucleotides encoding the AAV variants of the AAV library according to claim 1.

10. A library of vectors comprising the polynucleotides encoding the AAV variants of the AAV library according to claim 1.

11. A library of cloning cells comprising the AAV variants of the AAV library according to claim 1 and/or comprising polynucleotides encoding the same.

12. A method of generating an AAV library, comprising: a) generating variant capsid protein genes encoding variant of native AAV8 or AAV9 capsid proteins, the variant comprises a substituted sequence relative to native AAV 8 or AAV9 capsid protein, the substituted amino acid sequence is located at VR VIII region of SEQ ID NO:1 (AAV8) or SEQ ID NO:43 (AAV9); b) cloning said variant capsid protein genes into AAV vectors, wherein said AAV vectors are replication competent AAV vectors.

13. The method of claim 12, wherein VR VIII region is the position amino acids 585 to 597 or 598 of SEQ ID NO:1 (AAV8) or the position amino acids 583 to 595 or 596 of SEQ ID NO:43 (AAV9).

14. The method of claim 13, further comprising: 1) screening said AAV vector library from b) for variant AAV capsid proteins for increased transduction or tropism in human tissue or cells as compared to a non-variant parent capsid protein; and 2) selecting said variant AAV capsid vector from c).

15. Use of an AAV library according to claim 1, a method according to any one of claim 12, a library of polynucleotides according to claim 8, a library of vectors according to claim 10 and/or a library of cloning cells according to claim 9 for identifying an AAV variant infecting a target cell or tissue of interest.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1 shows the outline of in vivo screen strategy.

[0103] FIG. 2 shows the screen results. A) The week 1 screen result for liver. B) The week 1 screen result for brain. C) The week 4 screen result for various tissues. The result of starting library was marked in blue line.

[0104] FIG. 3 shows the effect of AAV8-VR VIII variants. A) Luciferase expression in HEK293T cells transduced with AAV8 and AAV8-VR VIII variants. MOI =10,000, n=3. B) In vivo luciferase expression in C57BL/6J mice 3 days after 1×10{circumflex over ( )}10 vg of control AAV8 and AAV8-VR VIII variants following intravenous injection. Negative control, PBS injected animals. The same results were observed in two independent biological repeats. C) Luciferase quantification of AAV8 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 3, 7 and 14. n=6 Data are reported as mean±SEM. D) Luciferase quantification of AAV8 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 3. E) Luciferase quantification of AAV8 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 7. F) Luciferase quantification of AAV8 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 14.The same results were observed in two independent biological repeats.

[0105] FIG. 4 shows at week 2 post injection, lung, liver, spleen, heart, kidney, lymph node, right quadriceps (LQ), left quadriceps (LQ) muscle and brain were harvested to detect the vector genome copy number in each tissue, n=6. The absolute GCNs in different tissues were plotted together for AAV8 (A), AAV8-Lib20 (B), AAV8-Lib25 (C), AAV8-Lib43 (D), AAV8-Lib44 (E), AAV8-Lib45 (F). The same results were observed in two independent biological repeats.

[0106] FIG. 5 shows the liver GCNs among different AAV8 VR VIII variants. Data are reported as mean±SEM

[0107] FIG. 6 shows at week 2 post injection, we determined the serum alanine transaminase (ALT) level. No significant change was noticed between control (PBS and AAV8) and AAV8-VR VIII variants.

[0108] FIG. 7 shows the effect of AAV9-VR VIII variants. A) In vivo luciferase expression in C57BL/6J mice 7 days after 1×10{circumflex over ( )}11 vg of control AAV9 and AAV9-VR VIII variants following intravenous injection. Negative control, PBS injected animals. B) Luciferase quantification of AAV9 and AAV9-VR VIII variants in C57BL/6J animals or PBS control. Data are reported as mean±SEM C) Luciferase expression and quantification (D) of AAV9 and AAV9-VR VIII variants in the head of C57BL/6J animals or PBS control. n=6 Data are reported as mean±SEM E) The head/body ratio of AAV9 and AAV9-VR VIII variants. For all the above experiments, the same results were observed in two independent biological repeats.

[0109] FIG. 8 shows luciferase expression in HEK293T cells transduced with AAV9 and AAV9-VR VIII variants. MOI=10,000, n=3.

[0110] FIG. 9 shows at week 2 post injection, tissues were harvested to detect the vector genome copy number, n=6. The absolute GCNs in each tissues were plotted for liver (A), brain (B), heart (C), and Lung (D). The same results were observed in two independent biological repeats.

[0111] FIG. 10 shows ALT level following AAV9 VR VIII variants-mediated gene delivery.

[0112] FIG. 11 shows the effect of AAV2-VR VIII variants. A) Luciferase expression in HEK293T cells transduced with AAV2 and AAV2-VR VIII variants. MOI=10,000, n=3. B) In vivo luciferase expression in C57BL/6J mice 3 days after 1×10{circumflex over ( )}10vg of control AAV2 and AAV2-VR VIII variants following intravenous injection. Negative control, PBS injected animals. The same results were observed in two independent biological repeats. C) Luciferase quantification of AAV2 and AAV2-VR VIII variants in C57BL/6J animals or PBS control at day 3, 7 and 14. n=6 Data are reported as mean±SEM.

[0113] FIG. 12 shows the effect of AAV2-VR VIII variants. A) In vivo luciferase expression in C57BL/6J mice 7 days after 1×10{circumflex over ( )}10 vg of control AAV2 and AAV2-VR VIII variants following intravenous injection. Negative control, PBS injected animals. B) Luciferase quantification of AAV2 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 7. C) In vivo luciferase expression in C57BL/6J mice 14 days after 1×10{circumflex over ( )}10 vg of control AAV2 and AAV2-VR VIII variants following intravenous injection. Negative control, PBS injected animals. D) Luciferase quantification of AAV8 and AAV8-VR VIII variants in C57BL/6J animals or PBS control at day 14. The same results were observed in two independent biological repeats.

[0114] FIG. 13 shows hFIX expression in monkey plasma.

DETAILED DESCRIPTION

[0115] The following description of the disclosure is merely intended to illustrate various embodiments of the disclosure. As such, the specific modifications discussed are not to be construed as limitations on the scope of the disclosure. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is understood that such equivalent embodiments are to be included herein. All references cited herein, including publications, patents and patent applications are incorporated herein by reference in their entirety.

[0116] The articles “a”, “an”, and “the” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “a polypeptide complex” means one polypeptide complex or more than one polypeptide complex.

[0117] As used herein, the term “about” or “approximately” refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1%to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 15%, 10%, 5%, or 1%.

[0118] Throughout this disclosure, unless the context requires otherwise, the words “comprise”, “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

Pharmaceutical Composition

[0119] The present disclosure also provides a pharmaceutical composition comprising the polypeptide complex or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.

[0120] The term “pharmaceutically acceptable” indicates that the designated carrier, vehicle, diluent, excipient (s) , and/or salt is generally chemically and/or physically compatible with the other ingredients comprising the formulation, and physiologically compatible with the recipient thereof.

[0121] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is bioactivity acceptable and nontoxic to a subject. Pharmaceutical acceptable carriers for use in the pharmaceutical compositions disclosed herein may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, or non-toxic auxiliary substances, other components known in the art, or various combinations thereof.

Method of Treatment

[0122] Therapeutic methods are also provided, comprising: administering a therapeutically effective amount of the polypeptide complex or the bispecific polypeptide complex provided herein to a subject in need thereof, thereby treating or preventing a condition or a disorder. In certain embodiments, the subject has been identified as having a disorder or condition likely to respond to the polypeptide complex or the bispecific polypeptide complex provided herein.

[0123] As used herein, the term “subject” includes any human or nonhuman animal. The term “nonhuman animal” includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.

[0124] The terms “treatment” and “therapeutic method” refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder.

[0125] In certain embodiments, the conditions and disorders include tumors and cancers, for example, non-small cell lung cancer, small cell lung cancer, renal cell cancer, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric carcinoma, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer, thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymic carcinoma, leukemia, lymphomas, myelomas, mycoses fungoids, merkel cell cancer, and other hematologic malignancies, such as classical Hodgkin lymphoma (CHL) , primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich B-cell lymphoma, EBV-positive and -negative PTLD, and EBV-associated diffuse large B-cell lymphoma (DLBCL) , plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma, and HHV8-associated primary effusion lymphoma, Hodgkin's lymphoma, neoplasm of the central nervous system (CNS) , such as primary CNS lymphoma, spinal axis tumor, brain stem glioma.

EXAMPLES

Example 1

The Equipments and Regents

[0126]

TABLE-US-00003 TABLE 1 The equipment used in the invention Equipments Product number Supplier SpectraMax ® M5/M5e SpectraMax ® Molecular Multimode M5/M5e Devices Plate Reader Diagnostica Stago STart ST art Diagnostica 4 Hemostasis Analyzer Stago EnVision 2105 2105-0010 PerkinElmer multimode plate reader Ice machine ST150 Sciencetool CYRO Vessel CY50935-70 Thermo Fisher Locator 4 PLUS Scientific    4° C. refrigerator HYC390F Haier −20° C. refrigerator DW-40L348 Haier −80° C. refrigerator 8960086V Thermo Fisher Scientific Biosafety cabinet BSC-II-A2 Sujing Incubator HERAcell 240i Thermo Fisher Scientific Countess ™ II AMQAX1000 Thermo Fisher cell counter Scientific Inverted Microscope ECLIPS T52 Nikon Refrigerated centrifuge 5424R Eppendorf Centrifuge 5810R Eppendorf Ultracentrifuge Optima XPN-100 Beckman Coulter Basic Power Supply PowerPac Basic Bio-Rad Amersham Imager 680 Amersham Imager GE Healthcare blot and gel imager 680QC NanoDrop One NanoDrop One/One.sup.c Invitrogen Microvolume UV-Vis UV-Vis Spectrophotometers Applied Biosystems QuantStudio ™ 7 Applied QuantStudio ™ 7 Biosystems Flex Real-Time PCR System ProFlex ™ 3 × 32- ProFlex ™ 3 × 32- Thermo Fisher well PCR well PCR Scientific System System-4484073 Tanon 2500/2500R 2500 R Tanon Gel Imaging System Milli-Q ® Direct 8 Water Direct 8 EMD Millipore Purification System

TABLE-US-00004 TABLE 2 The regents and supplies used in the study Reagents & Supplies Cell culture DMEM, High Glucose Gibco 11965118 HEK293T ATCC CRL-3216 ™ Trypsin-EDTA (0.25%), phenol red Invitrogen 25200072 DPBS Corning 21-031-CV FBS Corning 35-081-CV DMSO Sigma-Aldrich D2650 Antibiotic-Antimycotic, 100X Gibco 15240062 Countess ™ Cell Counting Chamber Slides Invitrogen C10312 Corning ® 150 mm TC-treated Culture Corning 430599 Dish 1.5 mL MaxyClear Snaplock Axygen Met-150-C Microcentrifuge Tube Construction of AAV NdeI NEB R0111S plasmid XbaI NEB R0145S NEBuilder HiFi DNA NEB E2621L Assembly Master Mix Ampicillin Sodium(100 mg/ml) TIANGEN RT501 Endura Competent Cells Lucigen 60241-2 0.2 mL Polypropylene PCR Tube Strips, Axygen PCR-0208-C 8 Tubes/Strip 8-Strip PCR Tube Caps for 0.2 mL PCR Axygen PCR-02CP-C Tube Strips, Clear PP AAV VR VIII variants PEI 25K Polysciences 23966-1 packaging, mixing for in EndoFree Plasmid Maxi Kit QIAGEN 12362 vivo selection & Benzonase Novagen 70664 Recombinant AAV OptiPrep ™ Density Gradient Medium Sigma-Aldrich D1556 packaging Power SYBR ™ Green PCR Master Mix Applied Biosystems 4367659 Fisherbrand ™ Cell Lifters Fisher Scientific 08100240 Quick-Seal Polypropylene Tube Beckman Coulter 342414 APOLLO 20 mL 150 KDa Concentrators Orbital Biosciences AP2015010 HiTrap ® Q High Performance GE Healthcare 17-1154-01 In vivo Selection for C57BL/6J mice Shanghai SLAC Laboratory liver-targeting variants Animal Co. DNesay Blood&Tissue kit QIAGEN 69506 NGS to quantify the Zymoclean ™ Gel DNA Recovery Kit Zymo Research D4002 AAV genome reads in Agarose Biowest 111860 tissues Marker II TIANGEN MD102 Gel Loading Dye, Purple (6X) NEB B7024S Titration of particles by AAV8 Titration ELISA PROGEN-PRAAV8 ELISA Recombinant Adeno-associated virus 8 ATCC VR-1816 In vivo rAAV-luciferase XenoLight D-Luciferin-K+ Salt PerkinElmer 122799-10 transduction and Bioluminescent Substrate detection ALT ActivityAssay Kit Sigma-Aldrich MAK052-1KT In vivo rAAV-hFIX F9 KO mice Shanghai Model Oranisms transduction Tissule, plasma and DPBS Corning serum collection 21-031-CV/Hyclone-5H30028.03 3.8% sodium citrate HIMEDIA-R014 10% Neutral buffered formalin INNOCHEM-A28231 Detection if hFIX VisuLize Factor IX Antigen Kit Affinity Biologicals FIX-AG expression Rox Factor IX Rossix 900020 In vivo viral genome Power SYBR ™ Green PCR Master Mix Applied Biosystems 4367659 copy number DEPC-Treated water Invitrogen AM9916 DNesay Blood&Tissue kit QIAGEN 69506 Hard-Shell ® 384-Well PCR Plates Bio-Rad H5P3801 Axygen ® 60 μm CyclerSeal Sealing Film Axygen PCR-TS for Storage and PCR Application Multiplate ™ 96-Well PCR Plates, low Bio-Rad MLP-9601 profile In vitro Infectivity Bright-Glo ™ Luciferase Assay System Promega E2620 96 Well Clear Round Bottom TC-Treated Corning 3799 Microplate Sodium dodecyl NuPAGE ™ 4-12% Bis-Tris Protein Gels, Invitrogen NP0321BOX sulfate-polyacrylamide 1.0 mm, 10-well NuPAGE ™ Sample Reducing Agent Invitrogen NP0009 (10X) Fast Silver Stain Kit Beyotime P0017S BenchMark ™ Protein Ladder Invitrogen 10747012

TABLE-US-00005 TABLE 3 The various oligos used in the study Construction of AAV plasmids pITR2-Rep2-Cap8- Forward 5′-TAAGCCAACTAGTGGAACCGGTGCGGCCGCACGCGTGGAGTTTAAGCCC library-ITR2-1 GAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCC GCCATGCCGGGGTT-3′ Reverse 5′-GAAGATAACCATCGGCAGCCATTTAATTAAACCTGATTTAAATCATTTA TTGTTCAAAG-3′ pITR2-Rep2-Cap8- Forward 5′-CTTTGAACAATAAATGATTTAAATCAGGTTTAATTAAATGGCTGCCGAT library-ITR2-2 GGTTAT CTTC-3′ Reverse 5′-TTCCAATTTGAGGAGCCGTGTTTTGCTGCTGCAACATATGGTTATCTGC CACGATACCGTATT-3′ pITR2-Rep2-Cap8- Forward 5′-ACACGGCTCCTCAAATTGGAATCTAGACTTGTCAACAGCCAGGGGGCCT library-ITR2-3 TACCCGGTATGGTCTG-3′ Reverse 5′-GCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTCGGTCCGCACGTGGT TACCTACAAAATGCTAGCTTACAGATTACGGGTGAGGTAACG-3′ Cap8-library Forward 5′-GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATG region pAAV-RC8 Forward 5′-TTGCTTGTTAATCAATAAACCG-3′ backbone Reverse 5′-ACCTGATTTAAATCATTTATTGTTCAAAGATGC-3′ pAAV-RC9 Forward 5′-TTGCTTGTTAATCAATAAACCG-3′ backbone Reverse 5′-ACCTGATTTAAATCATTTATTGTTCAAAGATGC-3′ Titering and Mixing of AAV Capsid Library Rep gene Forward 5′-GCAAGACCGGATGTTCAAAT-3′ Reverse 5′-CCTCAACCACGTGATCCTTT-3′ Titering of Recombinant AAV CMV promoter Forward 5′-TCCCATAGTAACGCCAATAGG-3′ Reverse 5′-CTTGGCATATGATACACTTGATG-3′ Forward 5′-TCCCATAGTAACGCCAATAGG-3′ Reverse 5′-CTTGGCATATGATACACTTGATG-3′ Next Generation Sequencing to quantify the AAV genome reads in tissues VR VIII region Forward 5′-CAAAATGCTGCCAGAGACAA-3′ Reverse 5′-GTCCGTGTGAGGAATCTTGG-3′ IN vivo viral genome copy number CMV promoter Forward 5′-TCCCATAGTAACGCCAATAGG-3′ Reverse 5′-CTTGGCATATGATACACTTGATG-3′

TABLE-US-00006 TABLE 4 The primers used for amplifying pAAV-RC9-library fragment1 target primers sequences Cap9-lib2-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib2-R AGTTTGTGTCTGGGGTGCAGTATTAGCCGATTGTAAGTTTGTGGCCA CTTGTCCATAGG Cap9-lib7-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib7-R GGTCCCTGTTTGAGGAGCGGTGTTTGCCGATTGCAGGTTTGTGGCCA CTTGTCCATAGG Cap9-lib31-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib31-R ATTTTCTGTAGTTGGACCAGTATTTGAGTTTTGCAAATTTGTGGCCA CTTGTCCATAGG Cap9-lib33-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib33-R AGTTCCGGTCGCAGGGGCTGTGTTGCTGCTCTGGAGATTTGTGGCCA CTTGTCCATAGG Cap9-lib43-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib43-R GGTCCCCGTTTGAGGAGCGGTGTTTGCCGACTGTAGGTTTGTGGCCA CTTGTCCATAGG Cap9-lib11-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib11-R AGTTCCTGTAGTTGGACCAGTGTTTGAGTTTTGCAAATTTGTGGCCA CTTGTCCATAGG Cap9-lib46-1 Cap9-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCT Cap9-lib46-R ATCTGCGGTAGCTGCTTGTGTGTTGCCGCTCTGGAGGTTTGTGGCCA CTTGTCCATAGG

TABLE-US-00007 TABLE 5 The primers used for amplifying pAAV-RC9-library fragment2 target primers sequences Cap9-lib2-2 Cap9-lib2-F AACTTACAATCGGCTAATACTGCACCCCAGACACAAACTGTTCAAAACC AAGGAATACTTC Cap9-R GTTTATTGATTAACAAGCAATTACAGATTACGAGTCAGGT Cap9-lib7-2 Cap9-lib7-F AACCTGCAATCGGCAAACACCGCTCCTCAAACAGGGACCGTTCAAAACC AAGGAATACTT Cap9-R GTTTATTGATTAACAAGCAATTACAGATTACGAGTCAGGT Cap9-lib31-2 Cap9-lib31-F AATTTGCAAAACTCAAATACTGGTCCAACTACAGAAAATGTTCAAAACC AAGGAATACTTC Cap9-R GTTTATTGATTAACAAGCAATTACAGATTACGAGTCAGGT Cap9-lib33-2 Cap9-lib33-F AATCTCCAGAGCAGCAACACAGCCCCTGCGACCGGAACTGTTCAAAACC AAGGAATACTT Cap9-R GTTTATTGATTAACAAGCAATTACAGATTACGAGTCAGGT Cap9-lib43-2 Cap9-lib43-F AACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGACCGTTCAAAACC AAGGAATACTT Cap9-R GTTTATTGATTAACAAGCAATTACAGATTACGAGTCAGGT Cap9-lib11-2 Lib-LP-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTT ATCT Cap9-R AGTTCCTGTAGTTGGACCAGTGTTTGAGTTTTGCAAATTTGTGGCCACT TGTCCATAGG Cap9-lib46-2 Lib-LP-F GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTT ATCT Cap9-R ATCTGCGGTAGCTGCTTGTGTGTTGCCGCTCTGGAGGTTTGTGGCCACT TGTCCATAGG

Example 2

Methods

Cell Culture

[0127] HEK293T cells were purchased from ATCC (ATCC, Manassas, Va.). HEK293T cells were maintained in complete medium containing DMEM (Gibco, Grand Island, N.Y.), 10% FBS (Corning, Manassas, Va.), 1% Anti-Anti (Gibco, Grand Island, N.Y.). HEK293T cells were grown in adherent culture using 15 cm dish (Corning, Corning, Calif.) in a humidified atmosphere at 37° C. in 5% CO.sub.2 and were sub-cultured after treatment with trypsin-EDTA (Gibco, Grand Island, N.Y.) for 2-5 min in the incubator, washed and re-suspended in the new complete medium.

Construction of AAV Plasmids

[0128] Plasmid pAAV-RC8 contains the Rep encoding sequences from AAV2 and Cap encoding sequences from AAV8. We generated a fragment that contains 5′ MluI and AAV's native promoter, upstream of the Rep2 gene in the pAAV-RC8 plasmid, by using the

TABLE-US-00008 forward primer: 5′-TAAGCCAACTAGTGGAACCGGTGCGGCCGCACGCGTGGAGTTTAAGC CCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGC AGCCGCCATGCCGGGGTT-3′, and reverse primer: 5′-GAAGATAACCATCGGCAGCCATTTAATTAAACCTGATTTAAATCATT TATTGTTCAAAG-3′.

[0129] To substitute VR VIII sequence of wild type AAV8, we introduced NdeI and XbaI restriction sites into 1756 bp and 1790 bp of the type 8 capsule (Cap8) gene, so the Cap8 region was generated by high-fidelity PCR amplification of two DNA fragments from plasmid pAAV-RC8.

[0130] One fragment was produced by using the

TABLE-US-00009 forward primer: 5′-CTTTGAACAATAAATGATTTAAATCAGGTTTAATTAAATGGCTGCC GATGGTTATCTTC-3′, and reverse primer: 5′-TTCCAATTTGAGGAGCCGTGTTTTGCTGCTGCAACATATGGTTATC TGCCACGATACCGTATT-3′; the other fragment was produced by using the forward primer: 5′-ACACGGCTCCTCAAATTGGAATCTAGACTGTCAACAGCCAGGGGGC CTTACCCGGTATGGTCTG-3′, and reverse primer: 5′-GCCAACTCCATCACTAGGGGTTCCTGCGGCCGCTCGGTCCGCACGT GGTTACCTACAAAATGCTAGCTTACAGATTACGGGTGAGGTAACG-3′.

[0131] Plasmid pssAAV-CMV-GFP-mut was digested by NotI (NEB, Ipswich, Mass.). The three fragments and linearized vector (pssAAV-CMV-GFP-mut) were assembled together with the NEB HiFi Builder (NEB, Ipswich, Mass.). The assembled product with the correct orientation and sequence was called pITR2-Rep2-Cap8-ITR2.

[0132] We then synthesized these 52 VR VIII oligo sequences with flanking 20nt overlapping sequences the same as Cap8 gene (Genewiz). These 52 sequences were used to substitute the VR VIII of AAV8 capsid backbone, individually, which were further subcloned into an all-in-one construct containing the modified capsid sequences with rep and inverted terminated repeats (ITRs) from AAV2 (FIG. 1A). Plasmid pITR2-Rep2-Cap8-ITR2 was digested with the enzyme NdeI (NEB, Ipswich, Mass.) and XbaI (NEB, Ipswich, Mass.) for linearization to generate a vector backbone. To substitute the wild type AAV8 VR VIII region, each VR VIII oligo was assembled with linearized pITR2-Rep2-Cap8-mut-ITR2 vector individually. The assembled product with the correct orientation and sequence was called pITR2-Rep2-Cap8-library-ITR2. Therefore, we have generated 52 different pITR2-Rep2-Cap8-library-ITR2 plasmids.

[0133] To generate recombinant pAAV-RC8-library plasmids, the whole Cap8-library fragment, 2.2 kb, from selected pITR2-Rep2-Cap8-library-ITR2 plasmids and backbone from pAAV-RC8, 5.2 kb, were assembled together using the NEB HiFi Builder (NEB, Ipswich, Mass.). Briefly, the whole Cap8-library region was produced by high-fidelity PCR amplification of plasmid pITR2-Rep2-Cap8-library-ITR2 using the forward primer 5′-GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCT-3′ and reverse primer 5′-GTTTATTGATTAACAAGCAATTACAGATTACGGGTGAGGT-3′. The vector backbone was produced by high-fidelity PCR amplification of plasmid pAAV-RC8 using the forward primer 5′-TTGCTTGTTAATCAATAAACCG-3′ and reverse primer 5′-ACCTGATTTAAATCATTTATTGTTCAAAGATGC-3′. The assembled product with the correct orientation and sequence was called pAAV-RC8-library.

[0134] Plasmid pAAV-RC9 contains the Rep encoding sequences from AAV2 and Cap encoding sequences from AAV9, synthesized by Genewiz. The whole Cap9-library region was produced by high-fidelity PCR amplification of two DNA fragments from plasmid pAAV-RC9. One fragment was produced by using the primer sets in the Table 4, the other fragment was produced by using the primer sets in the Table 5. The linear vector backbone of pAAV-RC9 was also produced by high-fidelity PCR amplification of plasmid pAAV-RC9 using the forward primer of 5′-TTGCTTGTTAATCAATAAACCG-3′ and reverse primer of 5′-ACCTGATTTAAATCATTTATTGTTCAAAGATGC-3′. The two DNA fragments and linearized vector (pAAV-RC9) were assembled together using NEB HiFi Builder (NEB, Ipswich, Mass.). The product with the correct orientation and sequence was called pAAV-RC9-library.

AAV Capsid Library Packaging

[0135] The packaging and purification of AAV capsid library were performed as previously described with some modifications. Briefly, HEK293T cells were co-transfected with 23.7 μg of individual pITR2-Rep2-Cap8-library-ITR2 plasmid and 38.7 μg of pHelper (Cell Biolabs) for separate packaging. Polyethyleneimine (PEI, linear, MW 25000, Polysciences, Inc., Warrington, Pa.) was used as transfection reagent. Cells were harvested 72 hrs post-transfection using cell lifter (Fisher Scientific, China), subjected to 3 rounds of freeze-thaw to recover the AAV variants inside the cells. The cell lysates were then digested with Benzonase (EMD Millipore, Denmark, Germany) and subjected to tittering by SYBR Green qPCR (Applied Biosystems, Woolston Warrington, UK) using primers specific to the Rep gene (forward: 5′-GCAAGACCGGATGTTCAAAT-3′, reverse: 5′-CCTCAACCACGTGATCCTTT-3′). 5×10.sup.9 vg of each AAV variants were then mixed together. The mixture was then purified on iodixanol gradient (Sigma, St. Louis, Mo.) in Quick-Seal Polypropylene Tube (Beckman Coulter, Brea, Calif.) followed by ion exchange chromatography using HiTrap Q HP (GE Healthcare, Piscataway, N.J.). The elution was concentrated by centrifugation using centrifugal spin concentrators with 150K molecular-weight cutoff (MWCO) (Orbital biosciences, Topsfield, Mass.). Following purification, the mixture containing 52 AAV VR VIII variants was quantified again by qPCR using the primer sets for Rep gene and diluted into two parts. The first part contains three independent aliquots acting as control viral mixture before selection. The second part was used for tail vein injection into C57BL/6J mice, at 2.5×10.sup.11 vg per animal, for in vivo selection.

[0136] When packaging rAAV-luciferase and rAAV-hFIX vectors, HEK293T cells were co-transfected with: i) pAAV-RC8 or selected pAAV-RC8-library and pAAV-RC9-library plasmids; ii) pAAV-CMV-Luciferase or pAAV-TTR-hFIX, respectively; iii) pHelper in equimolar amounts for each packaging. Plasmids were prepared using EndoFree Plasmid Kit (Qiagen, Hilder, Germany). The transfection, viral harvesting and purification steps were the same as the packaging of AAV VR VIII variants as mentioned above. The genome titer of the rAAV-luciferase vectors were quantified by qPCR using primers specific to the CMV promoter (forward: 5′-TCCCATAGTAACGCCAATAGG -3′, reverse: 5′-CTTGGCATATGATACACTTGATG -3′). The genome titer of the rAAV-hFIX vectors were quantified by qPCR using primers specific to the TTR promoter (forward: 5′-TCCCATAGTAACGCCAATAGG -3′, reverse: 5′-CTTGGCATATGATACACTTGATG-3′). The physical titer of rAAV8-and rAAV9-luciferase vectors were evaluated as described below (data not shown). The purity of rAAV were evaluated by SDS-PAGE silver staining, vector with ˜90% purity were used in our study (data not shown).

In Vivo Selection for Liver-Targeting Variants

[0137] All animal work was performed in accordance with institutional guidelines under the protocols approved by the institutional animal care and use committee of WuXi AppTec (Shanghai). The C57BL/6J mice (Shanghai SLAC Laboratory Animal Co., Ltd.), male, 6 to 8-week-old, were tail vein injected with mixture of AAV VR VIII variants as described above. At week 1, 2 and 4 post-injection, the animals were euthanized by cervical dislocation after being anesthetized with isoflurane. For week 1 and 2, liver and brain were harvested, and for week 4, lung, liver, spleen, heart, kidney, lymph node, quadriceps muscle and brain were also harvested. Then the total DNA was extracted using DNeasy Blood & Tissue Kit (QIAGEN) according to the manufacturer's protocol and then analyzed by next generation sequencing to compare the AAV read counts after selection vs before selection.

TABLE-US-00010 TABLE 7 The list of AAV8 VR VIII variants selected for further in vitro and in vivo validation. The variant name their VR VIII sequence in DNA and AA were showed. The mutations in reference to the VR VIII of AAV8 were marked in bold. Protein_seq (585-597, VP1 Variant name Coding_dna (1753-1791, VP1 numbering) numbering) SEQ ID NO WT AAV8 AACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAAC NLQQQNTAPQIGT  2 AAV8-Lib20 AACCTGCAATCGTCTACGGCCGGACCCCAGACACAGAC NLQSSTAGPQTQ 21 AAV8-Lib25 AACCTCCAGAGCGGCAACACACGAGCAGCTACCTCAG NLQSGNTRAATS 25 AAV8-Lib43 AACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGAC NLQSANTAPQTG  9 AAV8-Lib44 AATTTGCAAAACTCAAATACTGCTCCGAGTACTGGAAC NLQNSNTAPSTGT 37 AAV8-Lib45 AATTTCCAGAGCAGCAGCACAGACCCTGCGACCGGAG NFQSSSTDPATGD 38

TABLE-US-00011 TABLE 9 The list of AAV9 variants selected for further in vitro and in vivo validation. The variant name their VR VIII sequence in DNA and AA were showed. The mutations in reference to the VR VIII of AAV9 were marked in bold. Protein_seq (583-595, SEQ Variant name Coding_dna (1752-1791, VP1 numbering) VP1 numbering) ID NO WT AAV9 AACCACCAGAGTGCCCAAGCACAGGCGCAGACCGGCTGG NHQSAQAQAQTGW 33 AAV9-Lib2 AACTTACAATCGGCTAATACTGCACCCCAGACACAAACT NLQSANTAPQTQT  4 AAV9-Libll AATTTGCAAAACTCAAACACTGGTCCAACTACAGGAACT NLQNSNTGPTTGT 13 AAV9-Lib31 AATTTGCAAAACTCAAATACTGGTCCAACTACAGAAAAT NLQNSNTGPTTEN 29 AAV9-Lib33 AATCTCCAGAGCAGCAACACAGCCCCTGCGACCGGAACT NLQSSNTAPATGT 14 AAV9-Lib43 AACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGACC NLQSANTAPQTGT  9 AAV9-Lib46 AACCTCCAGAGCGGCAACACACAAGCAGCTACCGCAGAT NLQSGNTQAATAD 11

Next Generation Sequencing to Quantify the AAV Genome Reads in Tissues

[0138] The DNA from control viral mixture before injection and the total DNA isolated from various tissues were subjected to PCR to amplify the VR VIII region using the primer set (forward: 5′-CAAAATGCTGCCAGAGACAA-3′ and reverse: 5′-GTCCGTGTGAGGAATCTTGG-3′). The PCR products at the correct size were gel purified (Zymo Research, Irvine, Calif.) and then quantified by nanodrop. These products were analyzed by next generation sequencing with Illumina Hiseq X conducted at the WuXi NextCODE. During the analysis, the reads were separated by each VR VIII DNA sequence with no mismatch allowed. Then, we obtained the absolute read count of individual VR VIII in each experimental condition. Then, we converted the data into relative read count to normalize the difference for different time point and different tissues.

Titration of AAV Particles by ELISA

[0139] The AAV particle concentration was determined by the Progen AAV8 Titration ELISA kit (Progen Biotechnik GMBH, Heidelberg, Germany), against a standard curve prepared in the ELISA kit. Briefly, the recombinant adeno-associated virus 8 reference standard stock (rAAV8-RSS, ATCC, VR-1816) and samples were diluted with ready-to-use sample buffer so that they can be measured within the linear range of the ELISA (7.81×10.sup.6-5.00×10.sup.8 capsids/mL). The rAAV8-RSS was diluted in the range of 1:2000 to 1:16000, whereas samples were diluted between 1:2000 and 1:256000. Pipette 100 μL of ready-to-use sample buffer (blank), serial dilutions of standard, and samples (both diluted in ready-to-use sample buffer) into the wells of the microtiter strips. Seal strips with adhesion foil provided and incubate for 1 h at 37° C. Next, the plate was emptied and washed with 200 μL ready-to-use sample buffer 3 times. Pipette 100 μL biotin conjugate into the wells and seal strips with adhesion foil. After a 1-hour incubation at 37° C., the plates were emptied and washed 3 times. 100 μL streptavidin conjugate was then added to the wells and incubated for 1 hour at 37° C. Repeat washing step as described above, and pipette 100 μL substrate into the wells. Incubate the plate for 15 minutes at room temperature, and stop color reaction by adding 100 μL of stop solution into each well. Measure intensity of color reaction with a photometer at 450 nm wavelength within 30 minutes.

In Vitro Infectivity

[0140] HEK293T cells were seeded in 96-well cell-culture plates (Corning, Wujiang, J S) 16 hrs before transduction. Cells were mock infected or infected with rAAV-VR VIII variants individually, MOI=10,000, in serum- and antibiotic-free DMEM for 2 hrs. 48 hrs post infection, the cells were lysed to detect luciferase expression using the Bright-Glo™ Luciferase Assay System (Promega, Madison, Wis.) according to the manufacturer's instructions.

Sodium Dodecyl Sulfate-Polyacrylamide

[0141] For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, samples were denatured in NuPage Reducing Agent and NuPAGE LDS Sample Buffer (both from Invitrogen, Cartsbad, Calif.) at 100° C. for 10 min before being loaded onto NuPAGE 4-12% Bis-Tris minigels (Invitrogen, Cartsbad, Calif.). After electrophoresis, gels were silver-stained, using a Fast Silver Stain Kit (Beyotime, Shanghai, China). View gels using a white light box and a suitable imaging system.

In Vivo rAAV-Luciferase and Serum ALT Detection

[0142] The C57BL/6J mice, male, 6 to 8-week-old, were injected with appropriate amount of rAAV-luciferase vectors by tail vein injection. Bioluminescence were detected at day 3, week 1 and week 2 after viral injection. Before each detection, the mice will receive the 15 mg/ml D-Luciferin (PerkinElmer) by intraperitoneal injection. 10 mins after D-Luciferin injection, the mice will receive anesthesia using isoflurane. Xenogen Lumina II small animal in vivo imaging system (PerkinElmer) was used to select the region of interest (ROI), quantify and analyze the signal presented as photons/second/cm2/steridian (p/sec/cm2/sr). After the bioluminescence detection at week 2, the animals will be euthanized using 10% CO.sub.2 followed by serum and tissue collection for serum alanine aminotransferase (ALT) detection and genome copy number detection. The ALT levels was determined by Alanine Aminotransferase Activity Assay Kit (SIGMA) according to the manufacturer's protocol.

In Vivo rAAV-hFIX Transduction

[0143] The potency of rAAV-hFIX gene transfer efficiency was initially assessed in 6 to 8-week-old male wild-type C57BL/6J mice by assessing hFIX levels in plasma following tail vein injection of the vector. Next, the F9 KO mice in C57BL/6J background purchased from Shanghai Model Organisms, male, 6 to 8-week-old, were injected with appropriate amount of AAV vectors by tail vein injection to assess the efficacy.

Tissue, Plasma and Serum Collection

[0144] At appropriate time after viral injection, blood was collected by retroorbital bleeding. For terminal blood withdraw, immediately after CO.sub.2 euthanasia, cardiac puncture was performed to collect blood followed by perfusion using PBS to harvest livers. The largest liver lobe was fixed with 10% Neutral buffered formalin (NBF) for pathological examination. Two independent sampling of other liver lobes were collected for snap-frozen and maintained in −80° C. for genome copy number detection. For serum collection, blood is placed in 4° C. for 2 hrs. Then spin down the blood at 8000 rpm for 15 mins, and aspirate the supernatant. For plasma collection, blood was added into 3.8% sodium citrate at a ratio of 9:1. Then, spin down the mixture at 8000 rpm for 5 mins, and aspirate the supernatant. The serum and plasma were maintained in −80° C.

Detection of hFIX Expression and Activity

[0145] The hFIX expression level was determined by an enzyme-linked immunosorbent assay (ELISA) (Affinity Biologicals, Ancaster, ON, Canada) according to the manufacturer's protocol. Briefly, a flat-bottomed, 96-well plate was coated with goat antibody against human factor IX. Standards were made by using serial dilutions of calibrator plasma (0.0313-1 IU/mL). Mouse plasma was diluted 1:200 in sample diluent buffer, and 100 μL samples and standards were added to the wells. After a 1-hour incubation at room temperature, the plates were emptied and washed with 300 μL diluted wash buffer 3 times. The plates were then incubated for 30 minutes at r temperature with 100 μL horseradish peroxidase (HRP)-conjugated secondary antibody solution. After a final wash step, the HRP activity was measured with Tetramethylbenzidine (TMB) substrate. The color reaction was stopped after 10 minutes using stop solution and read spectrophotometrically at 450 nm within 30 minutes. The reference curve is a log-log plot of the absorbance values versus the factor IX concentration, and the factor IX content in plasma samples can be read from the reference curve.

[0146] The hFIX activity in mice was determined in a chromogenic assay using the ROX factor IX activity assay kit (Rossix, Mo{umlaut over ( )}lndal, Sweden) according to the manufacture's protocol. Briefly, standard dilutions were prepared using normal human plasma in diluent buffer, range from 25% to 200% activity (100% activity is defined as 1 IU/mL factor IX in plasma). The experimental plasma samples were diluted 1:320 in diluent buffer, and 25 μL samples and standards were added to low binding 96 well microplates. 25 μL Reagent A (containing lyophilized human factor VIII, human factor X, bovine factor V and a fibrin polymerization inhibitor) was added to the wells and incubated for 4 minutes at 37° C. And then 150 μL Reagent B (containing lyophilized human factor XIa, human factor II, calcium chloride and phospholipids) was added to the wells. After 8 minutes at 37° C., activated factor X generation was terminated by the addition of 50 μL factor Xa Substrate (Z-D-Arg-Gly-Arg-pNA), and the absorbance was read at 405 nm. Plot the maximal absorbance change/minute (ΔA405 max/min) vs. factor IX activity in a Log-Log graph, and the factor IX activity of the samples can be calculated using the reference curve.

In Vivo Viral Genome Copy Number

[0147] Absolute qPCR using SYBR Green (Applied Biosystems, Woolston Warrington, UK) was used to quantify AAV viral genome copy number. Total DNA was extracted from various tissues using DNeasy Blood & Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. Total DNA concentration was determined using Nanodrop, and 40ng of DNA from each sample was used as the template for qPCR. qPCR was performed on all tissue samples and control, done in triplicate, using primers specific for the CMV promoter (forward: TCCCATAGTAACGCCAATAGG, reverse: CTTGGCATATGATACACTTGATG). Linearized pssAAV-CMV-luci-mut plasmid at 2.89×10.sup.1, 2.89×10.sup.2, 2.89×10.sup.3, 2.89×10.sup.4, 2.89×10.sup.5, 2.89×10.sup.6, 2.89×10.sup.7 copy numbers (0.0002, 0.002, 0.02, 0.2, 2, 20, 200 pg) were used to generate a standard curve to calculate the copy numbers.

Example 3

In Vivo Selection

[0148] 52 different VR VIII sequences (Table 6). These 52 sequences were used to substitute the VR VIII of AAV8 capsid backbone, individually, which were further subcloned into an all-in-one construct containing the modified capsid sequences with rep and inverted terminated repeats (ITRs) from AAV2. These constructs were used to package wild-type-like AAV particles, individually, then mixed together at equal viral genome, followed by purification. The purified AAV variant library was divided into two parts. One part was used for NGS detection (n=3) as the starting library baseline. Another part was subjected to systemic delivery for in vivo selection to isolate liver and brain-targeting AAV variants (FIG. 1). Notably, the design contained all the available unique VR VIII sequences including that of WT AAV8, or AAV8-Lib40, in our screen (Table 6). We used AAV8-Lib40 as our internal control during the screening and selection process.

[0149] At week 1 post administration, liver and brain were harvested. Compared with the starting library and AAV8-Lib40, we were able to identify a few variants enriched in liver (FIG. 2A) and brain (FIG. 2B). At week 4 post administration, lung, liver, spleen, heart, kidney, lymph node, quadriceps (QA) muscle and brain were also harvested to evaluate biodistribution. We were able to identify variants that preferably target liver or brain than other tissues (FIG. 2C, Table 10).

TABLE-US-00012 TABLE 10 Variants that showed increased liver targeting, normalized to WT AAV8 to baseline as 100%. Protein_seq (585-597/8, SEQ Ratio (relative Variants VP1 numbering) ID No. wtAAV8) AAV8-Lib01 NNQNTNTAPTAGT  3 106.14% AAV8-Lib04 NNQAANTQAQTGL   6 227.97% AAV8-Lib05 NLQSGNTQAATSD  7 181.93% AAV8-Lib07 NLQSANTAPQTGT  9 230.06% AAV8-Lib08 NLQQTNSAPIVGA 10 116.07% AAV8-Lib09 NLQSGNTQAATAD 11 112.02% AAV8-Libll NLQNSNTGPTTGT 13 119.50% AAV8-Lib12 NLQSSNTAPATGT 14 253.36% AAV8-Lib13 NNQAANTQAQTGL  6 180.94% AAV8-Lib15 NNQSANTQAQTGL 16 257.83% AAV8-Lib19 NNQNATTAPITGN 20 239.08% AAV8-Lib20 NLQSSTAGPQTQT 21 181.06% AAV8-Lib21 NLQQQNTAPIVGA 22 150.83% AAV8-Lib23 NLQQTNSAPIVGA 10 110.46% AAV8-Lib25 NLQSGNTRAATSD 25 129.13% AAV8-Lib33 NLQSSNTAPATGT 14 100.38% AAV8-Lib35 NLQQQNTAPQIGT  2 131.97% AAV8-Lib36 NLQQTNTGPIVGN 32 121.99% AAV8-Lib37 NLQQTNTGPIVGN 32 131.34% AAV8-Lib38 NHQSAQAQAQTGW 33 121.67% AAV8-Lib40 NLQQQNTAPQIGT  2 100.00% AAV8-Lib43 NLQSANTAPQTGT  9 142.69% AAV8-Lib44 NLQNSNTAPSTGT 37 258.75% AAV8-Lib46 NLQSGNTQAATAD 11 111.35% AAV8-Lib47 NFQNNTTAADTEM 39 124.68% AAV8-Lib48 NLQSGNTQAATSD  7 176.81% AAV8-Lib49 NLQAANTAAQTQV 24 112.13% AAV8-Lib52 NLQQQNAAPIVGA 42 128.43%

[0150] While before the purification, we were able to titer all the AAV VR VIII variants individually for mixing equal amount and purification, we failed to detect AAV8-Lib26 by NGS both in our starting library and screens (FIG. 2A-C). This implied that the mutations in AAV8-Lib26 may not comply with the current AAV purification methods. Apart from this, we concluded that our capsid library design and screen strategy yielded highly viable AAV virions that facilitated the enrichment of liver- and brain-targeted variants.

[0151] To further validate the gene delivery capability, the selected VR VIII sequences (Table 7) were subcloned into recombinant AAV capsid plasmid for the packaging of luciferase reporter gene. AAV8-Lib25 and AAV8-Lib43 demonstrated significantly higher transgene expression in vitro (FIG. 3A). Importantly, we found most of novel AAV variants showed highly significant increase in in vivo transduction (FIG. 3B-3E). As negative control, AAV8-Lib45, whose VR VIII was downregulated during our screen, showed significantly decreased transduction both in vitro (FIG. 3A) and in vivo (FIGS. 3B and 3C). These results, to an extent, validated our screen process and results.

[0152] Furthermore, when we systemically characterized their biodistribution, we confirmed that these variants maintained a liver targeting profile as indicated by dominant GCNs in the liver than other tissues (FIG. 4A-E). Importantly, the liver genome copy numbers were significantly higher for AAV8 VR VIII variants than AAV8 (FIG. 5) further confirming the improved targeting capability. AAV8-Lib45, on the other hand, showed significantly lower liver GCNs further confirming our screen strategy (FIG. 5).

[0153] As a gene therapy vector, it is of most importance to have a good safety profile. To this end, we detected serum alanine transaminase (ALT) level, an important maker for liver toxicity. The ALT level was maintained below baseline for all of the groups (FIG. 6). These results indicate that AAV8 VRIIII variants could serve as alternative gene delivery tool to the liver.

[0154] As we have identified promising VR VIII sequences for gene delivery to the brain, we hypothesized that substituting WT VR VIII of AAV9 with brain-enriched VR VIII sequences (FIG. 2B) would generate variants with higher CNS-targeting capability. To test it, the AAV9-VR VIII capsid (Table 9) were used to package the genetic payload carrying luciferase reporter gene for evaluating transduction efficiency. We found that AAV9-Lib46 showed significantly higher transgene expression than WT AAV9 in vivo (FIGS. 7A and 7B). Interestingly, AAV9-Lib31, AAV9-Lib33, and in particular, AAV9-Lib43 showed a peripheral tissue-detargeting while maintained comparable CNS gene delivery (FIG. 7A). To this end, we specifically compare and qualify the luciferase expression in the head (FIGS. 7C and 7D) and found a dramatic shift for the head/body ratio of transgene expression (FIG. 7G).

[0155] Then, we tested the in vitro transduction of our leading candidates AAV9-Lib43 and AAV9-Lib46. Following infection HEK293T cells, AAV9-Lib43 showed significantly decreased transgene expression (FIG. 8) and AAV9-Lib46 showed significantly increased transgene expression (FIG. 8). These data were consistent with their overall body expression in vivo (FIGS. 7A and 7B).

[0156] Next, we profiled the biodistribution of AAV9 and AAV9 VR VIII variants. As is well known that AAV9 has a tropism for liver, heart and CNS, we observed significantly decreased GCNs in the liver for AAV9-Lib31, AAV9-Lib33, and AAV9-Lib43 and higher GCNs for AAV9-Lib46 (FIG. 9A), consistent with the transgene expression results (FIG. 8A). AAV9-Lib43 and AAV9-Lib46 demonstrated significantly increased GCNs in the brain (FIG. 9B). Though not significant, we also observed elevated GCNs in heart and lung (FIGS. 9C and 9D). Furthermore, no ALT elevation were detected following AAV9 VR VIII variants-mediated gene delivery (FIG. 10). These results indicate that AAV9 VRIIII variants could serve as alternative gene delivery tool to the CNS following systemic gene delivery.

Example 4

AAV2 VR VIII Variants

[0157] 5 sequences listed in Table 11 were used to substitute the VR VIII of AAV2 capsid backbone (corresponding to amino acid position 582-594 of WT AAV2 YP_680426.1 (GenBank: NC_001401.2), individually, which were further subcloned into an all-in-one construct containing the modified capsid sequences with rep and inverted terminated repeats (ITRs) from AAV2. These constructs were used to package wild-type-like AAV particles, individually, then mixed together at equal viral genome, followed by purification. The purified AAV variant library was divided into two parts. One part was used for NGS detection (n=3) as the starting library baseline. Another part was subjected to systemic delivery for in vivo selection to isolate liver and brain-targeting AAV variants.

[0158] To further validate the gene delivery capability, the selected VR VIII sequences (Table 11) were subcloned into recombinant AAV2 capsid plasmid for the packaging of luciferase reporter gene. AAV2-Lib20, AAV2-Lib25, AAV2-Lib43, AAV2-Lib44, AAV2-Lib45 demonstrated significantly lower transgene expression in vitro (FIG. 11A). Importantly, we found most of novel AAV variants showed highly significant decrease in in vivo transduction (FIG. 11B-11C and FIG. 12A-D).

TABLE-US-00013 TABLE 11 The list of AAV2 VR VIII variants selected for further in vitro and in vivo validation. The variant name their VR VIII sequence in DNA and AA were showed. The mutations in reference to the VR VIII of AAV2 were marked in bold. Variant Protein_seq (585-597, name Coding_dna (1753-1791, VP1 numbering) VP1 numbering) WT AAV8 AACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGAT NLQRGNRQAATAD AAV2-Lib20 AACCTGCAATCGTCTACGGCCGGACCCCAGACACAGACT NLQSSTAGPQTQT AAV2-Lib25 AACCTCCAGAGCGGCAACACACGAGCAGCTACCTCAGAT NLQSGNTRAATSD AAV2-Lib43 AACCTACAGTCGGCAAACACCGCTCCTCAAACGGGGACC NLQSANTAPQTGT AAV2-Lib44 AATTTGCAAAACTCAAATACTGCTCCGAGTACTGGAACT NLQNSNTAPSTGT AAV2-Lib45 AATTTCCAGAGCAGCAGCACAGACCCTGCGACCGGAGAT NFQSSSTDPATGD

Example 5

Delivering a Nucleic Acid Vector to a Cell and/Tissue Using rAAV Used to Package a Genetic Payload that Comprise a Heterologous Nucleic Acid Region Comprising a Sequence Encoding a Protein or Polypeptide of Interest

[0159] The protein or polypeptide of interest is a protein or polypeptide describe in Table 12-14.

[0160] AAV8-hFIX, AAV8-Lib25-hFIX and AAV8-Lib43-hFIX were injected into 3-4 yeas old male cynomolgus monkeys with the dose of 5E12 vg/kg, monkeys enrolled in these experiments were all tested with neutralization antibody titer<1:50 against AAV8. Blood samples were harvested before dosage and at Day3, week1, week2 and week3, hFIX expression were detected in plasma by ELISA. The result shows that all of AAV8, AAV-Lib25 and AAV8-Lib43 can express hFIX efficiently in monkeys, AAV8-Lib25 express higher hFIX than AAV8 and AAV8-Lib43 (FIG. 13).

TABLE-US-00014 TABLE 12 Exemplary Proteins and polypeptides of interest (Liver Disease) Non-limiting Exemplary diseases, Non-limiting Protein or disorders, NCBI Polypeptide or phenotypes Protein IDs Cystathionine-beta- Homocystinuria NP_000062.1, synthase (CBS) NP_001171479.1, NP_001171480.1 Factor IX (FIX) Hemophilia B NP_000124.1 Factor VIII (F8) Haemophilia A NP_000123.1, NP_063916.1 Glucose-6-phosphatase Glycogen Storage NP_001257326.1 catalytic subunit (G6PC) Disease Type I AAI30479.1 (GSD1) AAI36370.1 Glucose 6-phosphatase GSD-Ia NP_000142.2, (G6Pase) NP_001257326.1 Glucuronidase, MPSVII-Sly NP_000172.2, beta (GUSB) NP_001271219.1 Hemochromatosis Hemochromatosis NP_000401.1, (HFE) NP_620572.1, NP_620573.1, NP_620575.1, NP_620576.1, NP_620577.1, NP_620578.1, NP_620579.1, NP_620580.1 Iduronate 2-sulfatase MPSII-Hunter NP_000193.1, (IDS) NP_001160022.1, NP_006114.1 Iduronidase, alpha-1 MPSI-Hurler NP_000194.2, (IDUA) AAA51698.1 Low density lipoprotein Phenylketonuria NP_000518.1, receptor (LDLR) (PKU) NP_001182727.1, NP_001182728.1, NP_001182729.1, NP_001182732.1, AAP36025.1 Myophosphorylase McArdle disease NP_001158188.1, (PYGM) (glycogen storage NP_005600.1 disease type V, GSD5) N-acetylglucosam- Sanfilippo syndrome NP_000254.2 inidase, alpha (MPSIIIB) (NAGLU) N-sulfoglucosamine Mucopolysaccharidosis NP_000190.1 sulfohydrolase (SGSH) type NP_001339851.1 IIIA (MPS IIIA) NP 001339850.1 AAH47318.1 Ornithine OTC deficiency NP_000522.3, carbamoyltransferase AAA59975.1 (OTC) Phenylalanine Hypercholesterolaemia NP_000268.1 hydroxylase or Phenylketonuria (PAH) (PKU) UDP Crigler-Najjar NP_000454.1 glucuronosyltransferase syndrome 1 family, polypeptide A1 (UGT1A1)

TABLE-US-00015 TABLE 11 Exemplary Proteins and polypeptides of interest (CNS Disease) Non-limiting NCBI Non-limiting Exemplary diseases, Protein IDs or Patent Protein or Polypeptide disorders, or phenotypes SEQ ID NOs Acid alpha-glucosidase (GAA) Pompe disease NP_000143.2, NP_001073271.1, NP_001073272.1 ApaLI Mitochondrial heteroplasmy, YP_007161330.1 myoclonic epilepsy with ragged red fibers (MERRF) or mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) Aromatic L-amino acid Parkinson’s disease NP_000781.1, decarboxylase (AADC) NP_001076440.1, NP_001229815.1, NP_001229816.1, NP_001229817.1, NP_001229818.1, NP_001229819.1 Aspartoacylase (ASPA) Canavan’s disease NP_000040.1, NP_001121557.1 Battenin Ceroid lipofuscinosis NP_000077.1 neuronal 3 (CLN3) NP_001035897.1 NP_001273033.1 NP_001273034.1 NP_001273038.1 NP_001273039.1 AAH04433.1 Ceroid lipofuscinosis neuronal 2 (CLN2) Late infantile neuronal NP_000382.3, ceroidlipofuscinosis AAB80725.1 or Batten’s disease Cluster of Differentiation 86 Malignant melanoma NP_001193853.1, (CD86 or B7-2) NP_001193854.1, NP_008820.3, NP_787058.4, NP_795711.1 Cystathionine-beta-synthase (CBS) Homocystinuria NP_000062.1, NP_001171479.1, NP_001171480.1 Dystrophin or Minidystrophin Muscular dystrophy NP_000100.3, NP_003997.1, NP_004000.1, NP_004001.1, NP_004002.3, NP_004003.2, NP_004004.1, NP_004005.1, NP_004006.1, NP_004007.1, NP_004008.1, NP_004009.1, NP_004010.1, NP_004011.2, NP_004012.2, NP_004013.1, NP_004014.2 Frataxin (FXN) Friedreich ataxia (FA) NP_000135.2 NP_852090.1 AAH23633.1 AAH48097.1 Glial cell-derived Parkinson’s disease NP_000505.1, neurotrophic factor (GDNF) NP_001177397.1, NP_001177398.1, NP_001265027.1, NP_954701.1 Glutamate decarboxylase 1(GAD1) Parkinson’s disease NP_000808.2, NP_038473.2 Glutamate decarboxylase 2 (GAD2) Parkinson's disease NP_000809.1, NP_001127838.1 Hexosaminidase A, α polypeptide, also called Tay-Sachs NP_000511.2 beta-Hexosaminidase alpha (HEXA) Hexosaminidase B, β polypeptide, also called Tay-Sachs NP_000512.1, beta-Hexosaminidase beta (HEXB) NP_001278933.1 Interleukin 12 (IL-12) Malignant melanoma NP_000873.2, NP_002178.2 Methyl CpG binding protein 2 (MECP2) Rett syndrome NP_001104262.1, NP_004983.1 Myotubularin 1 (MTM1) X-linked myotubular myopathy NP_000243.1 NADH ubiquinone oxidoreductase subunit 4 Leber hereditary optic YP_003024035.1 (ND4) Nerve growth factor (NGF) Alzheimer’s disease NP_002497.2 neuropeptide Y (NPY) Parkinson’s disease, epilepsy NP_000896.1 Neurturin (NRTN) Parkinson’s disease NP_004549.1 Palmitoyl-protein thioesterase 1 (PPT1) Ceroid lipofuscinosis neuronal 1 NP_000301.1 (CLN1) AAH08426.1 Sarcoglycan alpha, beta, gamma, Muscular dystrophy SGCA delta, epsilon, or zeta NP_000014.1, (SGCA, SGCB, SGCG, NP_001129169.1 SGCD, SGCE, or SGCZ) SGCB NP_000223.1 SGCG NP_000222.1 SGCD NP_000328.2, NP_001121681.1, NP_758447.1 SGCE NP_001092870.1, NP_001092871.1, NP_003910.1 SGCZ NP_631906.2 Tumor necrosis factor receptor fused to an Arthritis, Rheumatoid arthritis SEQ ID NO. 1 of antibody Fc (TNFR:Fc) WO2013025079 Ubiquitin-protein ligase E3A (UBE3A) Angelman Syndrome (AS) NP_570853.1 NP_000453.2 NP_570854.1 NP_001341434.1 AAH02582.2 β-galactosidase 1 (GLB1) GM1 gangliosidosis NP_000395.3 AAB81350.1

TABLE-US-00016 TABLE 12 Exemplary Proteins and polypeptides of interest (Other Disease) Non-limiting Exemplary diseases, Non-limiting NCBI disorders, Protein IDs or Patent Protein or Polypeptide or phenotypes SEQ ID NOs Adenine nucleotide progressive external NP_001142.2 translocator (ANT-1) ophthalmoplegia Alpha-1-antitrypsin Hereditary NP_000286.3, (AAT) emphysema or NP_001002235.1, Alpha-1-antitrypsin NP_001002236.1, deficiency NP_001121172.1, NP_001121173.1, NP_001121174.1, NP_001121175.1, NP_001121176.1, NP_001121177.1, NP_001121178.1, NP_001121179.1, AAA51546.1, AAB59375.1 Aquaporin 1 (AQP1) Radiation Induced NP_932766.1 Xerostomia (RIX) NP_001126220.1 AAH22486.1 ATPase copper Menkes syndrome NP_000043.4 transporting NP_001269153.1 alpha (ATP7A) ATPase, Chronic heart failure NP_001672.1, Ca++ transporting, NP_733765.1 cardiac muscle, slow twitch 2 (SERCA2) C1 esterase Hereditary NP_000053.2 inhibitor (C1EI) Angioedema (HAE) AAH11171.1 AAB59387.1 AAA35613.1 Cyclic nucleotide Achromatopsia NP_001073347.1 gated channel alpha 3 (ACHM) AF272900.1 (CNGA3) AAH96300.1 AAI50602.1 Cyclic nucleotide Achromatopsia NP_061971.3 gated channel (ACHM) AAF86274.1 beta 3 (CNGB3) Cystic fibrosis Cystic fibrosis NP_000483.3 transmembrane conductance regulator (CFTR) Galactosidase, Fabry disease NP_000160.1 alpha (AGA) Glucocerebrosidase Gaucher disease NP_000148.2, (GC) NP_001005741.1, NP_001005742.1, NP_001165282.1, NP_001165283.1 Granulocyte- Prostate cancer NP_000749.2 macrophage colonystimulating factory (GM-CSF) HIV-1 gag-proΔrt HIV infection SEQ ID NOs. 1-5 of (tgAAC09) WO2006073496 Lipoprotein LPL deficiency NP_000228.1 lipase (LPL) Medium-chain Medium-chain NP_000007.1, acyl-CoA acyl-CoA NP_001120800.1, dehydrogenase dehydrogenase NP_001272971.1, (MCAD) (MCAD) deficiency NP_001272972.1, NP_001272973.1 Myosin 7A (MYO7A) Usher syndrome 1B NP_000251.3, NP_001120651.2, NP_001120652.1 Poly(A) binding Oculopharyngeal NP_000321.1 protein nuclear 1 Muscular Dystrophy (PABPN1) (OPMD) Propionyl CoA Propionic acidaemias NP_000273.2, carboxylase, NP_001121164.1, alpha polypeptide NP_001171475.1 (PCCA) Rab escort Choroideremia (CHM) NP_001138886.1 protein-1 (REP-1) NP_001307888.1 CAA55011.1 Retinal pigment Leber NP_000320.1 epithelium-specific congenital amaurosis protein 65kDa (RPE65) Retinoschisin 1 (RS1) X-Linked Retinitis NP_000321.1 Pigmentosa (XLRP) Short-chain acyl-CoA Short-chain acyl-CoA NP_000008.1 dehydrogenase dehydrogenase (SCAD) (SCAD) deficiency Very long-acyl-CoA Very long-chain NP_000009.1, dehydrogenase acyl-CoA NP_001029031.1, (VLCAD) dehydrogenase NP_001257376.1, (VLCAD) deficiency NP_001257377.1

[0161] The embodiments of the present invention have been described above, but the present invention is not limited thereto, and those skilled in the art can understand that modifications and changes can be made within the scope of the purport of the present invention. The manner of modifications and changes should fall within the scope of protection of the present invention.