Patent Application
20220403414
The present invention relates to gene therapy, especially refers to adeno-associated virus (AAV).
Clinical gene therapy has been increasingly successful owing both to an improving understanding of human disease and to breakthroughs in gene delivery technologies. Among these technologies, recombinant adeno-associated viral (rAAV) vectors are being used in a growing number of clinical trials. rAAV offers several important advantages: (1) Favorable safety profile: no known human disease is associated with AAV. The virus can only replicate under special circumstances. There is no viral protein in the vector. The rAAV vector rarely integrates into the host genome. (2) Ability to infect both dividing and non-dividing cells in vitro and in vivo. (3) Long-term expression of the transgene in various tissues. (4) Absence of enormous immune response. All of them contribute to proven efficacy in numerous animal models, as well as increasing number of clinical studies including Leber's congenital amaurosis, Hemophilia B, Alpha-1 antitrypsin deficiency, Parkinson's disease, Canavan's disease and muscular dystrophies. In 2012, the European Commission approved a rAAV-based product for lipoprotein lipase deficiency, the first gene therapy product approved by the western world. In 2017, voretigene neparvovec-rzyl (Luxturna), a gene therapy used to treat biallelic RPE65 mutations-associated retinal dystrophy marked the first US Food and Drug Administration (FDA) approved in vivo gene therapy product. On May 24, 2019, the US FDA approved the first drug for spinal muscular atrophy, which is also an AAV-based gene therapy.
Challenges of gene delivery using AAV vector arise from the simple consideration that the properties that allow AAV natural infection are distinct from those needed for most medical applications such as gene therapy. As a result, AAV vector engineering such that viruses can release from the constraints of natural evolution and thereby enable them to acquire novel and biomedically valuable phenotypes has been and will continue to be a major challenge in the research field.
With an increasing number of clinical stage gene therapy studies, AAV8 and AAV9, another two naturally-occurring serotypes, have demonstrated more powerful gene delivery capability. However, the primary cellular receptor for AAV8 and AAV9 remain unknown. At present, the engineering of AAV8 and AAV9 vectors for both basic understanding as well as gene delivery applications are limited.
The present invention provides a variant AAV capsid protein, comprising one or more amino acid substitutions, the capsid protein comprises a substituted amino acid sequence corresponding to VR VIII region of the native AAV 8 or AAV9 capsid protein.
In one embodiment, the variant AAV capsid protein comprises a variant AAV capsid protein comprising a substitution at one or more of amino acid residues N585, L586, Q587, Q588, Q589, N590, T591, A592, P593, Q594, 1595, G596, T597, V598, corresponding to amino acid sequence of the native AAV 8 (SEQ ID NO:1), the substitution of amino acid residues is selected from N585Y, L586N, L586Q, L586K, L586H, L586F, Q587N, Q588 N, Q588S, Q588A, Q588D, Q588G, Q589T, Q589A, Q589G, Q589S, Q589N, N590A, N590S, N590D, N590T, N590Q, T591S, T591A, T591R, T591E, T591G, A592Q, A592D, A592G, A592R, A592T, P593A, P593T, Q594T, Q594A, Q594I, Q594S, Q594D, I595A, I595T, I595V, I595T, I595S, I595Y, G596Q, G596S, G596A, G596E, T597A, T597L, T597D, T597S, T597N, T597V, T597W, T597M, V598D.
In one embodiment, the capsid protein comprise a substituted amino acid sequence at the amino acids corresponding to amino acid position 585 to 597 or 585 to 598 of the native AAV 8 (SEQ ID NO:1).
In one embodiment, the capsid protein comprise a substituted amino acid sequence of Formula I at the amino acids corresponding to amino acid position 585 to 598 of the native AAV 8 (SEQ ID NO:1): X1X2X3X4X5X6X7X8X9X10X11X12X13X14, wherein
X1 is selected from Asn and Tyr,
X2 is selected from Leu, Asn, Gln, Lys, His, and Phe,
X3 is selected RECTIFIED SHEET (RULE 91),
X4 is selected from Gln, Asn, Ser, Ala, Asp, and Gly,
X5 is selected from Gln, Thr, Ala, Gly, Ser, and Asn,
X6 is selected from Asn, Ala, Ser, Asp, Thr, and Gln,
X7 is selected from Thr, Ser, Ala, Arg, Glu, and Gly,
X8 is selected from Ala, Gln, Asp, Gly, Arg, and Thr,
X9 is selected from Pro, Ala, and Thr,
X10 is selected from Gln, Thr, Ala, Ile, Ser, and Asp,
X11 is selected from Ile, Ala, Thr, Val, Thr, Ser, and Tyr
X12 is selected from Gly, Gln, Ser, Ala, and Glu,
X13 is selected from Thr, Ala, Leu, Asp, Ser, Asn, Val, Trp, and Met,
X14 is selected from Val and Asp,
the sequence doesn't comprise an amino acids sequence of SEQ ID NO:2 (native AAV8 VR VIII).
In one embodiment, the capsid protein comprise a substituted amino acid sequence of Formula IV at the amino acids corresponding to amino acid position 585 to 597 of the native AAV 8 (SEQ ID NO:1): X1X2X3X4X5X6X7X8X9X10X11X12X13, wherein
X2 is selected from Leu, Asn, His, and Phe,
X4 is selected from Gln, Asn, Ser, and Ala,
X5 is selected from Gln, Thr, Ala, Gly, Ser, and Asn,
X6 is selected from Asn, Thr, and Gln,
X7 is selected from Thr, Ser, and Ala,
X8 is selected from Ala, Gln, Gly, and Arg,
X9 is selected from Pro and Ala,
X10 is selected from Gln, Thr, Ala, Ile, Ser, and Asp,
X11 is selected from Ile, Ala, Thr, and Val
X12 is selected from Gly, Gln, Ser, Ala, and Glu,
X13 is selected from Thr, Ala, Leu, Asp, Asn, Val, Trp, and Met,
the sequence doesn't comprise a amino acids sequence of SEQ ID NO:2 (native AAV8 VR VIII).
In one embodiment, the capsid protein comprise a substituted amino acid sequence of Formula II at the amino acids corresponding to amino acid position 585 to 597 of the native AAV 8 (SEQ ID NO:1): X1X2X3X4X5X6X7X8X9X10X11X12X13, wherein
X2 is selected from Leu, Asn, and Phe,
X4 is selected from Gln, Asn, Ser, and Ala,
X5 is selected from Thr, Ala, and Ser,
X6 is selected from Asn, Ser, and Thr,
X7 is selected from Thr, Ala, and Gly,
X8 is selected from Ala, Gln, Gly, and Arg,
X9 is selected from Pro and Ala,
X10 is selected from Gln, Ala, and Ile,
X11 is selected from Thr and Val,
X12 is selected from Gly and Gln,
X13 is selected from Thr, Leu, Asn, and Asp.
In the invention, the NCBI Reference Sequence of WT AAV8 capsid protein is YP_077180.1 (GenBank: AAN03857.1), as shown in SEQ ID NO:1.
In one specific embodiment, the present invention provides a variant AAV 8 capsid protein, comprising a substituted sequence corresponding to the position amino acids 585 to 597 of SEQ ID NO:1 (AAV8); preferably, the sequence comprises a amino acids sequence selected from the groups consisting of SEQ ID NO:3-42 as shown in Table 6, preferably selected from the groups consisting of SEQ ID NO: 2-3, 6-7, 9-11, 13-14, 16, 20-22, 24, 25, 32-33, 37, 39, 42 as shown in Table 10.
In one specific embodiment, the present invention provides a variant AAV 8 capsid protein, comprising a substituted sequence corresponding to the position amino acids 585 to 597 of SEQ ID NO:1 (AAV8); preferably, the sequence comprises a amino acids sequence selected from the groups consisting of SEQ ID NO:21 (AAV 8-Lib20), SEQ ID NO:25 (AAV 8-Lib25), SEQ ID NO:9 (AAV 8-Lib43), and SEQ ID NO:37 (AAV 8-Lib44).
In some embodiment, the AAV variant is AAV serotype 9. The present invention provides an AAV library comprising a multitude of adeno-associated virus (AAV) variants, the AAV variants comprises a variant AAV capsid protein comprising a substitution at one or more of amino acid residues N583, H584, Q585, 5586, A587, Q588, A589, Q590, A591, Q592, T593, G594, W595, V596, corresponding to amino acid sequence of the native AAV 9 (SEQ ID NO:43), the substitution of amino acid residues is selected from N583Y, H584N, H584Q, H584K, H584L, H584F, Q585N, S586N, S586Q, S586A, S586D, S586G, A587T, A587Q, A587G, A587S, A587N, Q588A, Q588S, Q588D, Q588T, Q588N, A589S, A 589T, A589R, A589E, A589G, Q590A, Q590D, Q590G, Q590R, Q590T, A591P, A591T, Q592T, Q592A, Q5921, Q592S, Q592D, T593A, T5931, T593V, T593S, T593Y, G594Q, G594S, G594A, G594E, W595A, W595L, W595D, W595S, W595N, W595V, W 595T, W595M, V596D.
In one embodiment, the present invention provides a variant AAV 9 capsid protein a substituted amino acid sequence corresponding to VR VIII region of the native AAV9 capsid protein. The capsid protein comprise a substituted amino acid sequence of Formula I at the amino acids corresponding to amino acid position 583 to 596 of the native AAV 9 (SEQ ID NO:43): X1X2X3X4X5X6X7X8X9X10X11X12X13X14, wherein
X1 is selected from Asn and Tyr,
X2 is selected from Leu, Asn, Gln, Lys, His, and Phe,
X3 is selected from Gln and Asn,
X4 is selected from Gln, Asn, Ser, Ala, Asp, and Gly,
X5 is selected from Gln, Thr, Ala, Gly, Ser, and Asn,
X6 is selected from Asn, Ala, Ser, Asp, Thr, and Gln,
X7 is selected from Thr, Ser, Ala, Arg, Glu, and Gly,
X8 is selected from Ala, Gln, Asp, Gly, Arg, and Thr,
X9 is selected from Pro, Ala, and Thr,
X10 is selected from Gln, Thr, Ala, Ile, Ser, and Asp,
X11 is selected RECTIFIED SHEET (RULE91); Val, Thr, Ser, and Tyr
X12 is selected from Gly, Gln, Ser, Ala, and Glu,
X13 is selected from Thr, Ala, Leu, Asp, Ser, Asn, Val, Trp, and Met,
X14 is selected from Val, and Asp,
the sequence doesn't comprise a amino acids sequence of SEQ ID NO:33 (native AAV9 VR VIII).
In one embodiment, VR VIII region is the position amino acids 583 to 595 of SEQ ID NO:43 (AAV9), as compared to a wild-type AAV9 capsid proteins; the capsid protein comprise a substituted amino acid sequence of Formula II at the amino acids corresponding to amino acid position 585 to 597 of the native AAV 9 (SEQ ID NO:43): X1X2X3X4X5X6X7X8X9X10X11X12X13, wherein
X5 is selected from Ala, Ser, and Gly,
X8 is selected from Ala, Gln, and Gly,
X10 is selected from Gln, Thr, and Ala,
X12 is selected from Gly, Gln, Ala, and Glu,
X13 is selected from Thr, Asn, and Asp.
In the invention, the NCBI Reference Sequence of WT AAV9 capsid protein is AAS99264.1 (GenBank: ANF53541.1), as shown in SEQ ID NO:43.
MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNA
ADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEA
AKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASG
GGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYF
GYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQV
FTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSY
EFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQ
QRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDA
DKVMITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPH
TDGNFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRW
NPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL
In one specific embodiment, the present invention provides a variant AAV 9 capsid protein, comprising a substituted sequence corresponding to the position amino acids 583 to 595 or 596 of SEQ ID NO:43 (AAV9); preferably, the sequence comprises a amino acids sequence selected from the groups consisting of SEQ ID NO: 3-42 as shown in Table 8.
In one specific embodiment, the present invention provides a variant AAV 9 capsid protein, comprising a substituted sequence corresponding to the position amino acids 583 to 595 of SEQ ID NO:43 (AAV9); preferably, the sequence comprises a amino acids sequence selected from the groups consisting of SEQ ID NO:29 (AAV 9-Lib31), SEQ ID NO:14 (AAV 9-Lib 33), SEQ ID NO:9 (AAV 9-Lib43), and SEQ ID NO:11 (AAV 9-Lib46).
In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence that encodes above variant AAV capsid protein or vectors comprising the above polynucleotides.
The present invention provides an isolated, genetically modified host cell comprising the above polynucleotide.
The present invention provides a recombinant AAV virion comprising above variant AAV capsid protein.
In one specific embodiment, the present invention provides a pharmaceutical composition comprising:
a) a recombinant adeno-associated virus virion disclosed in the present invention; and
b) a pharmaceutically acceptable excipient.
In another aspect, the present invention provides a recombinant AAV vector, comprising polynucleotide encoding a variant AAV capsid protein of invention, and an AAV 5′ inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional gene product, a regulatory sequence which directs expression of the gene product in a target cell, and an AAV 3′ ITR.
In one specific embodiment, the regulatory sequence further comprises at least of an enhancer, a promoter, intron, and poly A.
In another aspect, the present invention also provides a method of delivering a nucleic acid vector encoding a functional gene product to cells and/or tissues with a recombinant AAV virion or recombinant AAV vector of the present invention.
In one specific embodiment, the present invention also provides the use of a recombinant AAV virion or recombinant AAV vector of the present invention in the preparation of product for delivering a nucleic acid vector encoding a functional gene product to cells and/or tissues.
In another aspect, the present invention also provides a method of treating disease, the method comprising administering to a subject in need thereof an effective amount of a recombinant AAV virion of present invention, the recombinant AAV virion comprises functional gene product.
In one specific embodiment, the present invention also provides the use of a recombinant AAV virion or recombinant AAV vector of the present invention in the preparation of product for treating disease to a subject in need thereof; preferably, the disease is selected from the group consisting of liver disease, central nervous system diseases, and other diseases.
In one specific embodiment, the gene product is a polypeptide.
In one specific embodiment, the polypeptide is selected from the group consisting of Cystathionine-beta-synthase (CBS), Factor IX (FIX), Factor VIII (F8), Glucose-6-phosphatase catalytic subunit (G6PC), Glucose 6-phosphatase (G6Pase), Glucuronidase, beta (GUSB), Hemochromatosis (HFE), Iduronate 2-sulfatase (IDS), Iduronidase, alpha-1 (IDUA), Low density lipoprotein receptor (LDLR), Myophosphorylase (PYGM), N-acetylglucosaminidase, alpha (NAGLU), N-sulfoglucosamine sulfohydrolase (SGSH), Ornithine carbamoyltransferase (OTC), Phenylalanine hydroxylase (PAH), UDP glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1); preferably wherein the recombinant AAV virion comprises a variant AAV8 capsid protein comprising a amino acids sequence selected from the groups consisting of SEQ ID NO: 2-3, 6-7, 9-11, 13-14, 16, 20-22, 24, 25, 32-33, 37, 39, 42 as shown in Table 10, preferably SEQ ID NO:21 (AAV8-Lib20), SEQ ID NO:25 (AAV8-Lib25), SEQ ID NO:9 (AAV8-Lib43), and SEQ ID NO:37 (AAV8-Lib44), and comprises a variant AAV9 capsid protein comprising a amino acids sequence of SEQ ID NO:11 (AAV9-Lib46).
In one specific embodiment, the polypeptide is selected from the group consisting of Acid alpha-glucosidase (GAA), ApaL1, Aromatic L-amino acid decarboxylase (AADC), Aspartoacylase (ASPA), Battenin, Ceroid lipofuscinosis neuronal 2 (CLN2), Cluster of Differentiation 86 (CD86 or B7-2), Cystathionine-beta-synthase (CBS), Dystrophin or Mini dystrophin, Frataxin (FXN), Glial cell-derived neurotrophic factor (GDNF), Glutamate decarboxylase 1 (GAD1), Glutamate decarboxylase 2 (GAD2), Hexosaminidase A, α polypeptide, also called beta-Hexosaminidase alpha (HEXA), Hexosaminidase B, β polypeptide, also called beta-Hexosaminidase beta (HEXB), Interleukin 12 (IL-12), Methyl CpG binding protein 2 (MECP2), Myotubularin 1 (MTM1), NADH ubiquinone oxidoreductase subunit 4 (ND4), Nerve growth factor (NGF), neuropeptide Y (NPY), Neurturin (NRTN), Palmitoyl-protein thioesterase 1 (PPT1), Sarcoglycan alpha, beta, gamma, delta, epsilon, or zeta (SGCA, SGCB, SGCG, SGCD, SGCE, or SGCZ), Tumor necrosis factor receptor fused to an antibody Fc (TNFR:Fc), Ubiquitin-protein ligase E3A (UBE3A), β-galactosidase 1 (GLB1); preferably wherein the recombinant AAV virion comprises a variant AAV9 capsid protein comprising a amino acids sequence selected from the groups consisting of SEQ ID NOs:9 (AAV9-Lib43) and SEQ ID NOs:11 (AAV9-Lib46).
In one specific embodiment, the polypeptide is selected from the group consisting of Adenine nucleotide translocator (ANT-1), Alpha-1-antitrypsin (AAT), Aquaporin 1 (AQP1), ATPase copper transporting alpha (ATP7A), ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 (SERCA2), C1 esterase inhibitor (ClEI), Cyclic nucleotide gated channel alpha 3 (CNGA3), Cyclic nucleotide gated channel beta 3 (CNGB3), Cystic fibrosis transmembrane conductance regulator (CFTR), Galactosidase, alpha (AGA), Glucocerebrosidase (GC), Granulocyte-macrophage colonystimulating factory (GM-CSF), HIV-1 gag-proArt (tgAAC09), Lipoprotein lipase (LPL), Medium-chain acyl-CoA dehydrogenase (MCAD), Myosin 7A (MYO7A), Poly(A) binding protein nuclear 1 (PABPN1), Propionyl CoA carboxylase, alpha polypeptide (PCCA), Rab escort protein-1 (REP-1), Retinal pigment epithelium-specific protein 65 kDa (RPE65), Retinoschisin 1 (RS1), Short-chain acyl-CoA dehydrogenase (SCAD), Very long-acyl-CoA dehydrogenase (VLCAD).
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.
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.
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%.
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.
The present disclosure also provides a pharmaceutical composition comprising the polypeptide complex or the bispecific polypeptide complex provided herein and a pharmaceutically acceptable carrier.
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.
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.
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.
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.
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.
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.
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% CO2 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.
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 forward primer:
To substitute VR VIII sequence of wild type AAV8, we introduced Ndel and Xbal 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.
One fragment was produced by using the forward primer:
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.
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 (
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′-GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTAT CT-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.
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.
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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′-CCTCAACCACGTGAT CCTTT-3′). 5×109 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 2.5×1011 vg per animal, for in vivo selection.
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 was used in our study (data not shown).
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.
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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.
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 (7.81×106-5.00×108 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.
HEK293T cells were seeded in 96-well cell-culture plates (Corning, Wujiang, JS) 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.
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
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% CO2 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
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.
At appropriate time after viral injection, blood was collected by retroorbital bleeding. For terminal blood withdraw, immediately after CO2 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
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.
The hFIX activity in mice was determined in a chromogenic assay using the ROX factor IX activity assay kit (Rossix, Molndal, 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 (ΔA405max/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.
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 40 ng 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 2.89×101, 2.89×102, 2.89×103, 2.89×104, 2.89×105, 2.89×106, 2.89×107 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.
52 different VR VIII sequences (Table 6) their corresponding GenBank ID were summarized in Table 7. 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 (
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 (
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 (
To further validate the gene delivery capability, the selected VR VIII sequences (Table 8) 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 (
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 (
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 (
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 (
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 (
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 (
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.
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 (
The protein or polypeptide of interest is a protein or polypeptide describe in Table 12-14.
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 (
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.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2019/111525 | Oct 2019 | CN | national |
This application claims the benefit of and priority to PCT Application No. PCT/CN2019/111525, filed Oct. 16, 2019, the entire contents of which are incorporated by reference herein for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/121099 | 10/15/2020 | WO |
