Lentiviral vector for treating human immunodeficiency virus infection

Abstract

Provided herein are a vector for eradicating human immunodeficiency virus (HIV) from a human comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV. Methods of treating of HIV and kits are also provided.

Description

Inventor: Laura A. PrendergastLENTIVIRAL VECTOR FOR TREATING HUMAN IMMUNODEFICIENCY VIRUS INFECTIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international patent application filed in accordance with the Patent Cooperation Treaty. This application claims the priority benefit of U.S. Provisional Patent Application No. 63 / 681,804, filed 10 August 2024, and entitled “LENTIVIRAL VECTOR FOR TREATING HUMAN IMMUNODEFICIENCY VIRUS INFECTION”. The disclosure of U.S. Provisional Patent Application No. 63 / 681,804 is hereby incorporated herein by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (VCT-0001- PV_SequenceListingFinal_10August2024.xml; Size: 391,000 bytes; Date of Creation: August 10, 2024) are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0003] The present invention generally relates to lentiviral vectors, compositions, and methods for preventing, treating, and / or eliminating human immunodeficiency virus (HIV) infection.BACKGROUND

[0004] Human immunodeficiency virus (HIV) infection / acquired immunodeficiency syndrome (HIV / AIDS) is a disease affecting primarily cells of the human immune system caused by infection with HIV. AIDS has caused over 30 million deaths and continues to present a global health crisis with close to 40 million people infected with HIV worldwide.

[0005] While HIV / AIDS is now considered to be a chronic disease in the developed world, without treatment survival time is estimated to be around 8-10 years. Combination antiretroviral therapy has made HIV a chronic manageable disease but is not a cure. HIV DNA remains latent, or inactive, in host reservoirs. Host reservoirs are immune system cells that are infected by HIV but are not actively producing new virus. HIV can hide in these cells for years and at some time become active again. Patients must continue treatment and sometimes change treatments to address when treatments become ineffective.SUMMARY

[0006] Current treatments for HIV do not address virus sequestered in latent cells; may cause side effects; require continued treatment; and often fail over time, requiring the prescribing ofInventor: Laura A. Prendergast new antiretroviral (ARV) drug regimens. The inventor has recognized the need for treatments that can eliminate HIV from a patient and obviate the need for continued treatment and the side effects associated with current treatments. The inventor has discovered, in various embodiments, a lentiviral gene delivery vehicle, a lentiviral vector, that can be administered to a subject that will effectively eradicate or eliminate HIV from the subject. In various embodiments, provided herein is an inducible, replication-competent, conditionally autoinhibiting lentiviral vector for eradicating human immunodeficiency virus (HIV) from infected cells in humans. In some embodiments, described herein is a vector for eradicating human immunodeficiency virus (HIV) from a human, comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV- infected cells and represses replication of the vector in the absence of HIV. In various embodiments described herein, the vector for eradicating human immunodeficiency virus (HIV) from a human is a lentiviral vector. In some embodiments described herein, the lentiviral vector is a feline immunodeficiency virus (FlV)-based vector. In some embodiments described herein the FIV-based vector comprises a modified wild type FIV strand and an FIV-based cargo strand.

[0007] The lentiviral gene delivery vehicle that can be administered to a subject to effectively eradicate or eliminate HIV from a subject is referred to throughout this application in various ways and is alternatively described as “lentiviral vector”, “FIV-based lentivector,” “(FIV)- based vector”, “FIV-based vector”, “lentivector”, “delivery vector,” “delivery vehicle” and “vector” and based on the context, persons of skill in the art would understand that these terms are, in the context of this disclosure, equivalent.

[0008] Provided, in various embodiments, is a lentiviral vector that provides an ‘eradicating’ or ‘sterilizing’ cure, wherein the HIV virus is removed from all cells harboring the virus. Eradication of HIV is defined as the complete elimination of HIV from the body, including the excision of HIV sequences from the subject’s genome.

[0009] The lentiviral vector described herein, in various embodiments is an inducible replication-competent conditionally auto-inhibiting lentiviral vector, which solves the problem of HIV infection by delivering genetic material that inhibits the HIV virus from budding out of infected cells thereby allowing infected cells to enzymatically eliminate HIV virions through endogenous digestive pathways. Because the lentiviral vector described herein is replication-competent and integrates into host cells, both dividing and non-dividing,Inventor: Laura A. Prendergast the vectors, compositions, methods, and kits described herein can provide for a cure of HIV from HIV-infected persons in need of treatment after a single administration.

[0010] In some embodiments, provided herein is an FIV vector, or delivery vector, that encodes a dual-function protein (2STOP) that prevents infectious HIV from budding from membranes of infected cells and represses replication of the lentiviral delivery vector under conditions once HIV virions have been eradicated from infected cells. That is, where HIV virions have been eradicated, the replication of the lentiviral delivery vector is repressed. The FIV-based vector, or delivery vector, is referred to herein in various embodiments as a vector for eradicating human immunodeficiency virus (HIV) from a human. In various embodiments, the inducible, replication-competent, conditionally auto-inhibiting lentiviral vector for eradicating human immunodeficiency virus (HIV) from infected cells in humans, is packaged in a feline immunodeficiency virus (FIV) coat.

[0011] 2STOP is a dual function protein that, in various embodiments, comprises a domain with a dominant-negative function that inhibits infectious HIV from budding out of the membrane of HIV-infected cells and a transcriptional repressor domain for repressing vector replication. Provided herein is a vector for eradicating human immunodeficiency virus (HIV) from a human, comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV. In some embodiments, the vector for eradicating human immunodeficiency virus (HIV) from a human is a lentiviral vector. In some embodiments, the lentiviral vector is a feline immunodeficiency virus (FlV)-based vector. In some embodiments, the FIV-based vector comprises a modified wild type FIV strand and an FIV cargo strand. In some embodiments, the FIV cargo strand comprises the nucleic acid sequence encoding the dual-function protein which inhibits infectious HIV from budding out of HIV-infected cells and represses replication of the vector in the absence of HIV. In some embodiments, the nucleic acid sequence on the FIV cargo strand that encodes the dual function protein is 2STOP.

[0012] In various embodiments, provided herein is a vector for eradicating human immunodeficiency virus (HIV) from a human, comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV, wherein the dual -function protein is 2STOP. In various embodiments, 2STOP comprises a transcriptional repressor domain that represses vector replication, and a dominant negative domain that inhibits budding of HIVInventor: Laura A. Prendergast from membranes of HIV infected cells. In various embodiments, the protein 2STOP is 2STOPA and is encoded by a nucleic acid sequence designated 2STOPA and wherein the sequence encoding the transcriptional repressor domain of 2STOPA is 5’ of the sequence encoding the dominant negative domain. In various embodiments, the nucleic acid encoding 2STOPA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents thereof. In various embodiments, the amino acid sequence of 2STOPA is selected from a group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and functional equivalents thereof. In some embodiments, 2STOPA comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and functional equivalents thereof.

[0013] In various embodiments, the protein 2STOP is 2STOPB and is encoded by a nucleic acid sequence designated 2STOPB and wherein the sequence encoding the transcriptional repressor domain of 2STOPB is 5’ of the sequence encoding the dominant negative domain. In various embodiments, the nucleic acid encoding 2STOPB is encoded by a nucleic acid sequence designated 2STOPB, and wherein the nucleic acid sequence encoding the dominant negative domain of 2STOPB is 5’ of the sequence encoding the transcriptional repressor domain. In various embodiments, the nucleic acid sequence designated 2STOPB is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and functional equivalents thereof. In various embodiments, the amino acid sequence of 2STOPB is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 and functional equivalents thereof. In various embodiments, 2STOPB comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to any one of the amino acid sequences set forth in a group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQInventor: Laura A. PrendergastID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and functional equivalents thereof.

[0014] In some embodiments, the domain for inhibiting budding of HIV from membranes of HIV infected cells is encoded by a sequence designated DN-lirl. In some embodiments, the sequence designated DN-lirl is SEQ ID NO: 45. In some embodiments, the amino acid sequence of DN-lirl is set forth in SEQ ID NO: 46 In various embodiments, DN-lirl comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98%, or 99%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46.

[0015] In some embodiments, the transcription repressor domain of 2STOP is selected from a group consisting of Gal4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR, and functional equivalents. In some embodiments, the transcriptional repressor domain amino acid sequence is selected from a group consisting of SEQ ID NO: 47, SEQ ID NO: 48 SEQ ID NO: 49, SEQ ID NO: 50 SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and functional equivalents.

[0016] In some embodiments, the 2STOP coding sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents thereof, and is downstream of a Transactivation Response Region (TAR sequence) SEQ ID NO: 69. The TAR sequence is an HIV-encoded sequence recognized by HIV’s transactivator molecule (tat), delivered early in the process of HIV infection.Interaction of tat with the TAR sequence results in upregulation of TAR-1 ab eled coding sequences.

[0017] In some embodiments, the genetic material coding for the 2STOP is delivered in vivo by means of a lentivector. In some embodiments, the lentivector is feline immunodeficiency virus (FIV).

[0018] In some embodiments, the FIV-based lentivector comprises a modified wild-type strand and a cargo strand to accomplish in vivo delivery of the 2STOP gene.

[0019] In some embodiments, the modified FIV wild-type strand comprises an operator sequence to accomplish inhibition of vector replication. In some embodiments, the operator sequence is situated downstream from the native FIV 5 ’Long Terminal Repeat (5’ LTR) which controls replication of the FIV genome and mediates expression of FIV genes.Inventor: Laura A. Prendergast

[0020] In various embodiments the cargo strand comprises an operator sequence for transcriptional repression of downstream sequences. In some embodiments, the operator sequence on the cargo strand is identical to the operator sequence on the modified wild-type strand.

[0021] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human, the dual-function 2STOP protein comprises a cognate transcriptional repressor wherein the operator sequence on the modified wild-type strand and on the cargo strand is recognized and bound by the cognate transcriptional repressor.

[0022] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human, the operator sequence on the modified wild-type strand and on the cargo strand is selected from a group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and functional equivalents thereof.

[0023] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human, the cargo strand further comprises a constitutive promoter operably linked to the 2STOP coding sequence. In some embodiments, the constitutive promoter that is operably linked for expression of 2STOP is selected from the group consisting of hCMV, EFlalpha, CAGG and SV40 and functional equivalents thereof. In some embodiments the constitutive promoter operably linked to the 2STOP coding sequence is selected from: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, and SEQ ID NO: 84 and functional equivalents thereof.

[0024] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human, replication of the lentivector is placed under the control of a cell-type specific promoter sequence. Cell-type specific promoters are selected from the group consisting of CD4, CD8, and CD34, and functional equivalents thereof. In some embodiments, the cell-type specific promoter is selected from the group consisting of CD4 (SEQ ID NO: 85), CD8 (SEQ ID NO: 86), CD34 (SEQ ID NO: 87), and functional equivalents.

[0025] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human the modified FIV wild-type strand comprises a 5’ Long Terminal Repeat (LTR) SEQ ID NO: 88, a 3’ LTR SEQ ID NO: 89, a primer binding site (PBS), SEQ ID NO: 90, and a sequence SEQ ID NO: 91 for packaging the lentivector genomic strand into the FIV shell.Inventor: Laura A. Prendergast

[0026] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human the FIV cargo strand further comprises adjunctive protective transgenes. In various embodiments, adjunctive protective transgenes encode multi-mechanistic approaches to attacking the HIV virus. In some embodiments, adjunctive protective transgenes include nucleic acid aptamers, protein aptamers, short hairpin RNA (shRNA), immunologic agents, and / or CRISPR-based gene editing technology for knockout of HIV gene products or excision of HIV sequences from the genome.

[0027] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human the adjunctive protective transgenes comprise one or more nucleic acid sequences coding for gene-editing technology. In some embodiments, the gene editing technology comprises Transcriptional Activator Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based editing, Zinc Finger Nucleases (ZFNs), and functional equivalents.

[0028] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human the gene-editing technology is CRISPR-based comprising one or more CRISPR-associated proteins (Cas proteins) and one or more signal guide RNAs (sgRNA) for site-specific gene editing. In some embodiments, the nucleic acid sequences coding for the Cas proteins to be delivered are selected from a group consisting of saCas9 SEQ ID NO: 92 or cjCas9 SEQ ID NO: 93 sagCas9 SEQ ID NO: 94, or Cas(I> SEQ ID NO: 95 and functional equivalents thereof.

[0029] In some embodiments, the amino acid sequences of the Cas proteins are selected from a group consisting of saCas9 SEQ ID NO: 96 or cjCas9 SEQ ID NO: 97 sagCas9 SEQ ID NO: 98, or Cas SEQ ID NO: 99, and functional equivalents thereof.

[0030] In some embodiments, the CRISPR-based gene editing technology comprises one or more sgRNA sequences that direct site-specific insertion of the FIV-based vector genome into “safe harbor” loci in the recipient’s genome to avert insertional mutagenesis or other positional effects. In some embodiments, sgRNA sequences for directing insertion of the vector into safe harbor loci are selected from SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and the like.

[0031] In some embodiments, the CRISPR-based gene editing technology comprises one or more sgRNA sequences that direct site-specific insertion of the FIV-based vector genome intoInventor: Laura A. PrendergastHIV coding sequences to accomplish gene knockout. In some embodiments, the HIV coding sequences targeted for gene knockout are gag-pol, integrase, env, vif, vpu, vpr, rev, vpx, and the like. Persons of skill in the art would understand that insertion of vector sequences into HIV coding sequences should be assessed in an ex vivo system for individual subjects to identify and protect against insertional mutagenesis or other positional effects in an in vivo system for treatment purposes.

[0032] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s gag-pol sequence are selected from a list that includes SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114,SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119,SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124,SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, and the like.

[0033] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s integrase sequence to accomplish gene knockout are selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and the like.

[0034] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s env sequence are selected from SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and the like.

[0035] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vif sequence are selected from SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159 and the like.

[0036] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpu sequence are selected from SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, and the like.

[0037] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpr sequence are selected from a list that includes SEQ ID NO: 169, SEQInventor: Laura A. PrendergastID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, and the like.

[0038] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s rev sequence are selected from SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the like.

[0039] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpx sequence are selected from SEQ ID NO: 193, SEQ ID NO: 194, and the like.

[0040] In some embodiments of the vector for eradicating human immunodeficiency virus (HIV) from a human, the CRISPR-based gene-editing technology directs insertion of the FIV-based vector genome into the human CCR5 T-cell receptor to accomplish a gene knockout of HIV’s preferred mode of entry into the human T-cells. In some embodiments, the sgRNA sequences directing insertion of the FIV-based vector into the human CCR5 T- cell receptor are selected from a list that includes SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, and the like.

[0041] In some embodiments, the CRISPR-based gene editing technology comprises one or more sgRNA sequences that direct excision of HIV sequences from the genome of infected host cells, thereby eliminating the latent reservoir of HIV-infected cells. See, for example, Ebina H, Misawa N, Kanemura Y, Koyanagi Y. Harnessing the CRISPR / Cas9 system to disrupt latent HI V-l provirus. Sci Rep. 2013; 3:2510.

[0042] In some embodiments, the adjunctive protective transgenes further comprise shRNA sequences for targeted knockout of expressed HIV genes. In some embodiments, the HIV gene products selected for knockout by shRNA are selected from a group consisting of gag- pol, integrase, env, vif, vpu, vpr, rev, vpx combinations thereof.

[0043] In some embodiments, the shRNA sequences for gag-pol knockout are selected from a list that includes SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218,SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223,SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228,Inventor: Laura A. PrendergastSEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, and the like.

[0044] In some embodiments, the shRNA sequences for integrase knockout are selected from a list that includes SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, and the like.

[0045] In some embodiments, the shRNA sequences for env knockout are selected from a list that includes SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, and the like.

[0046] In some embodiments the shRNA sequences for vif knockout are selected from a list that includes SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, and the like.

[0047] In some embodiments, the shRNA sequences for vpu knockout are selected SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, and the like.

[0048] In some embodiments, the shRNA sequences for vpr knockout are selected from SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, and the like.

[0049] In some embodiments, the shRNA sequences for rev knockout are selected from SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQ ID NO: 275, SEQID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQ ID NO: 280, SEQID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQ ID NO: 285, and the like.

[0050] In some embodiments, the shRNA sequences for vpx knockout are selected from SEQ ID NO: 286, SEQ ID NO: 287, SEQ ID NO: 288, SEQ ID NO: 289, SEQ ID NO: 290, SEQ ID NO: 291, SEQ ID NO: 292, SEQ ID NO: 293, SEQ ID NO: 294, SEQ ID NO: 295 and combinations thereof.

[0050] In various embodiments, the vector for eradicating human immunodeficiency virus (HIV) can further comprise adjunctive protective transgenes for targeting other viruses, such as, for example: Herpes simplex virus 1, Herpes simplex virus 2, Varicella-zoster virus, Epstein-Barr virus, Cytomegalovirus, Human herpesvirus 6, Human herpesvirus 7, Kaposi’s sarcoma-associated herpesvirus, Vaccinia virus, Variola virus, Monkeypox virus, CowpoxInventor: Laura A. Prendergast virus, Orf virus, Molluscum contagiosum virus, Hepatitis B virus, Woodchuck hepatitis virus, Duck hepatitis B virus, African swine fever virus, and the like.

[0051] In some embodiments, the vector for eradicating human immunodeficiency virus (HIV) comprises adjunctive protective transgenes which encode immunologic agents for targeting HIV virions in an infected host. Wherein the immunologic agent is selected from the group consisting of monoclonal antibodies (mAbs), broadly neutralizing monoclonal antibodies (bNAbs), monovalent antibodies, antigen-binding fragments (Fabs), and camelid nanobodies. Additional antibody formats include bispecific or trispecific antibodies, fusion antibodies (e.g., CD4-Env fusion proteins), anti -idiotypic antibodies, Fc-engineered antibodies with enhanced effector function or extended half-life, functional equivalents, and combinations thereof.

[0052] In certain embodiments, additional immunologic-based strategies are employed to target HIV-infected cells or modulate the host immune response. Such strategies include cytotoxic lymphocyte (CTL)-based therapies, including ex vivo expansion and adoptive transfer of HIV-specific CD8+T cells; chimeric antigen receptor (CAR)-modified immune cell therapies, including CAR-T and CAR-NK cells; therapeutic vaccination approaches designed to elicit or boost durable HIV-specific cellular and humoral immunity; immune checkpoint blockade (e.g., anti-PD-1, anti-CTLA-4) to restore T-cell function in exhausted HIV-specific populations; cytokine-based modulation to enhance antiviral effector activity (e.g., IL-2, IL-7, IL-15, IFN-a); and antibody-derived effector molecules such as immunotoxins, antibody-drug conjugates, or enzyme conjugates directed against HIV-infected cells. These immunologic interventions may be used individually or in combination with antibody -based strategies to augment viral clearance and improve functional cure outcomes.

[0053] In some embodiments, the immunologic agents for targeting HIV virions in an infected host comprise anti -idiotypic antibodies configured to stimulate the host’s immune system to generate endogenous anti-HIV antibodies. Such anti -idiotypic antibodies mimic one or more antigenic determinants of HIV and thereby elicit an adaptive immunologic response capable of recognizing and neutralizing HIV virions (See e.g., Kelley, B., et al. Human anti-idiotypic monoclonal antibody to the antigen-binding site of an anti-gp!20 antibody induces HIV- 1 -neutralizing immune responses. AIDS Research and Human Retroviruses. 1992;8(9): 1539-1544).Inventor: Laura A. Prendergast

[0054] In some embodiments, the present disclosure provides adjunctive protective transgenes encoding nucleic acid aptamers or protein aptamers that specifically bind to one or more structural or functional components of HIV virions. Such aptamers may be configured to interfere with viral entry, replication, or assembly by sterically hindering interactions between viral proteins and their corresponding host-cell receptors, or by directly disrupting essential viral enzymatic functions. Expression of these aptamers within an infected host cell facilitates targeted inhibition of HIV replication and contributes to suppression or clearance of the infection (See e.g., Zhou, J., Rossi, J. Cell-type-specific, aptamer-shRNA delivery: progress in cancer therapeutics. Expert Opinion on Drug Delivery. 2014; 11(12): 1781-1789).

[0056] In some embodiments, the FIV cargo strand further comprises auxiliary transgenes for targeting circulating anti -HIV antibodies specific to the strain of HIV that is infecting the host to accomplish seroreconversion. Such auxiliary transgenes may encode, without limitation, nucleic acid aptamers, protein aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, or camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents; and combinations thereof.

[0057] In some embodiments, the lentivector bearing the anti-HIV transgenes is administered as an adjuvant to immunologic-based approaches to neutralizing HIV that are exogenously administered. Such immunologic-based treatments include, for example: monoclonal antibodies (mAbs), monovalent antibodies (mAbs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bispecific antibodies, engineered immuno-adhesins, functional equivalents and combinations thereof.

[0058] In some embodiments, the FIV cargo strand further comprises nucleic acid sequences encoding a heterologous envelope glycoprotein for pseudo-typing the lentivector to provide additional host tropism. In some embodiments, the heterologous envelope glycoproteins are taken from a virus selected from the group consisting of, but not limited to, Vesicular Stomatitis Virus (VSV-G), Lymphocytic choriomeningitis virus (LCMV), Alphavirus Ross River virus (RRV), Marburg virus, Lassa virus, Baculovirus, functional equivalents, and combinations thereof.

[0059] In certain embodiments, FIV-based lentiviral vectors are pseudotyped with heterologous envelope glycoproteins to confer broad or tissue-specific tropism. ExemplaryInventor: Laura A. Prendergast glycoproteins include VSV-G (SEQ ID NO: 302), which enables high -titer production and transduction of nearly all mammalian cells; LCMV-GP (SEQ ID NO: 303), which confers neurotropism and affinity for hepatocytes, dendritic cells, macrophages, lymphocytes, and CNS -associated structures; RRV-Env (SEQ ID NO: 304), which targets muscle, joint, and connective tissue; MARV-GP (SEQ ID NO: 305), which directs entry into hepatocytes, endothelial cells, macrophages, and dendritic cells, enabling multi-organ targeting; LASV-GP (SEQ ID NO: 306), which exhibits broad tropism for liver, APCs, endothelium, and multiple visceral organs; and GP64 (SEQ ID NO: 307), which mediates broad, low-cytotoxicity transduction of hepatocytes, endothelial, fibroblast, neuronal, and respiratory epithelial cells, permitting repeated dosing. Such pseudotyping enables delivery to dividing and non-dividing cells and can be tailored for systemic or localized gene transfer applications.

[0060] In some embodiments, the present disclosure provides a method for in vivo eradication of HIV in an HIV-infected subject, comprising administering an effective dose of a vector comprising a nucleic acid sequence encoding a dual-function protein. The dual-function protein is configured to (i) repress vector replication through auto-inhibition in the absence of HIV and (ii) inhibit the budding of infectious HIV particles from the membranes of HIV-infected cells. In certain embodiments, the effective dose is determined by performing a T-cell count on the subject to calculate the required number of transducing units, wherein the lentiviral vector is administered at a multiplicity of infection (MOI) of about 1 to 50 per T-cell. In various embodiments, the vector can be delivered at concentrations greater than or equal to 103, 104, 105, 106, 107, 108, 109, IO10, or 1011transducing units (TU) / ml.Determination of therapeutically effective dosages is within the purview of a person of ordinary skill in the art.

[0061] In some embodiments, the vector is administered intravenously. In some embodiments, the vector is introduced intraarterially, intralesionally, percutaneously, subcutaneously, intramuscular, intrathecally, intraorbitally, intradermally, intraperitoneally, transtracheally, subcuticularly, by intrastemal injection, by inhalation or intranasal spraying, by endrotracheal route, or combinations thereof.

[0062] In some embodiments, the method for in vivo eradication of HIV in an HIV-infected subject includes determining the specific strain or clade of HIV infecting the subject, thereby enabling personalized or strain-specific therapeutic targeting. Strain identification may be accomplished using one or more diagnostic techniques, including enzyme-linked immunosorbent assays (ELISAs), Western blot analysis, and reverse transcription polymeraseInventor: Laura A. Prendergast chain reaction (RT-PCR), or combinations thereof. In certain embodiments, the kit comprises strain-specific targeting tools such as primers, reagents, and clade-specific standards configured for use in RT-PCR, enabling the detection and discrimination of distinct HIV clades or subtypes. The kit may further include calibrated reference materials suitable for clinical diagnostics or experimental applications, ensuring reproducibility and assay standardization. Based on the identified strain, the lentiviral vector may include one or more adjunctive protective transgenes or auxiliary transgenes selected to enhance therapeutic specificity and efficacy.

[0063] In various embodiments, the present disclosure provides a modular kit designed to support researchers and developers in the production, characterization, and deployment of lentiviral-based vectors, including those derived from feline immunodeficiency virus (FIV). The kit is optimized for use in targeted gene delivery applications.

[0064] In some embodiments, the kit comprises vials, tubes, or other containers bearing essential elements of the lentiviral vector genome which can be transported in a cooler on ice or on dry ice. In some embodiments, the kit further comprises vials containing buffers, enzymes, and nucleic acids appropriate for cloning of genetic material encoding anti-HIV transgenes into the lentiviral vector genomic construct.

[0065] In some embodiments, the kit comprises one or more containers — such as vials, tubes, or other storage vessels — housing genetic material encoding anti-HIV transgenes suitable for incorporation into the vector. The anti-HIV transgenes may be selected from a group comprising nucleic acid aptamers, protein aptamers, broadly neutralizing monoclonal antibodies (bNAbs), monoclonal antibodies (mAbs), monovalent antibodies (MAbs), antigenbinding fragments (Fabs), camelid-derived nanobodies, single-guide RNAs (sgRNAs) for CRISPR-based genome editing, short hairpin RNAs (shRNAs) for targeted gene silencing of HIV sequences, and combinations thereof. In certain embodiments, the kit further comprises genetic material encoding anti -idiotypic antibodies or related immunologic agents designed to elicit an anti-HIV antibody response in the host. In additional embodiments, the kit may include components specifically configured to target circulating anti-HIV antibodies in order to facilitate seroreconversion.

[0066] In some embodiments, the present disclosure provides a kit comprising a lentiviral- based gene delivery system that includes one or more nucleic acid constructs customizable to encode therapeutic or experimental payloads. In certain embodiments, the vector encodes a therapeutic protein (“2STOP”) which is designed to inhibit HIV replication through a dualInventor: Laura A. Prendergast mechanism: by trapping nascent HIV virions at the host cell membrane during budding, and by providing feedback inhibition that suppresses replication of the vector itself. The lentiviral-based system may further comprise envelope protein pseudotyping modules selected to enable receptor-specific targeting of mammalian cells, thereby conferring tissue or cell-type specificity to the resulting vector. In various embodiments, the system also includes regulatory elements that are activated in the presence of HIV infection — such as HIV- responsive transcriptional switches — together with auto-inhibition modules configured to limit off-target expression or uncontrolled vector propagation. Additionally, the system may optionally comprise wild-type FIV-derived nucleic acid strands, enabling the study of replication competence and latency mechanisms associated with lentiviral vectors.

[0067] In some embodiments, the kit comprises strain-specific targeting tools that include primers, standards, and reagents suitable for use in reverse transcription polymerase chain reaction (RT-PCR). These reagents are configured to enable the detection and discrimination of specific HIV clades and subtypes, thereby supporting personalized or strain-tailored therapeutic strategies. The kit may further include calibrated reference materials formulated for use in clinical diagnostics or experimental research, thereby ensuring reproducibility, accuracy, and standardization across assay platforms.

[0068] In some embodiments, the kit includes transfection and production reagents configured to support efficient lentiviral vector generation. These may comprise high- efficiency transfection reagents and plasmid systems optimized for lentiviral packaging. The kit may further include media formulations that are compatible with lentiviral vector production in mammalian cell lines, thereby facilitating robust and scalable vector yields. In certain embodiments, the kit provides optional packaging cell lines, including but not limited to HEK293T cells or feline-derived cell lines, which are specifically optimized for pseudotyping with feline immunodeficiency virus (FlV)-based envelope or core components.

[0069] In some embodiments, the kit comprises quality control and biosafety modules designed to evaluate the performance and safety of lentiviral vectors produced using the system. These modules may include both functional and physical titration assays for quantifying vector potency and determining viral particle concentration. The kit may further comprise molecular markers and analytical assays for assessing integration efficiency and verifying successful transduction events in target cells. Additionally, the kit may provide standardized biosafety protocols, containment guidelines, and operational procedures to support safe handling and regulatory compliance in research or clinical production settings.Inventor: Laura A. Prendergast

[0070] In some embodiments, the kit includes administration components suitable for the delivery of lentiviral vectors in preclinical models. In various embodiments the kit comprises needles, catheters, syringes, or related devices for administration of the vector into a living subject. These components may comprise sterile delivery instruments configured for intravenous (IV), intramuscular (IM), or intrathecal administration, depending on the intended route of delivery.

[0071] In some embodiments, the vector can be administered by various routes selected from a list that includes: bone marrow, into joints, intravenous, intra-arterial, intracranial intramuscular, subcutaneous, into various organs, intra-tumor, into the interstitial spaces, intra-peritoneal, intralymphatic, or into a capillary bed, and the like. The kit may also include buffer solutions and cry opreservation media for vector formulation and storage, as well as reconstitution instructions to ensure proper preparation and handling of the vector prior to administration.

[0072] In some embodiments, the kit further comprises documentation and regulatory support materials to facilitate compliance with manufacturing and clinical standards. These materials may include Good Manufacturing Practice (GMP) traceability documentation such as Certificates of Analysis (CoAs), Safety Data Sheets (SDS), and production batch records. Additionally, the kit may provide template documents for informed consent, investigational drug protocols, and regulatory submissions, thereby supporting the translation of lentiviral - based therapies into patient-directed clinical applications.

[0073] In some embodiments, the kit comprises instructional materials that provide guidance for the proper use of the kit components. The instructions may include a description of the FIV-based vector system and, optionally, descriptions of additional components provided in the kit. In certain embodiments, the instructions further comprise methods for determining the HIV strain infecting the subject, as well as protocols for selecting an appropriate dosage and route of administration. These instructional materials may be provided in printed form, electronically, or both, and are intended to facilitate effective and consistent application of the kit in research or therapeutic contexts.

[0074] In some embodiments, other infectious viruses can be targeted by means of a vector with an analogous genetic regulatory mechanism as described herein. In some embodiments, other infectious viruses that might be targeted by means of a vector with an analogous genetic regulatory mechanism as described herein include enveloped viruses. Examples of enveloped viruses that can be targeted in this manner can be selected from a list that includes DNAInventor: Laura A. Prendergast viruses such as Herpesviridae, Poxviridae, Hepadnaviridae, and Asfarviridae; and RNA viruses such as Flaviviridae, Alphaviridae, Togaviridae, Coronavirus, Hepatitis D, Orthomyxoviruses, Paramyxoviridae, Rhabdovirus, Bunyaviruses, and Filoviruses; Retroviruses, and the like.

[0075] In some embodiments, the vector described herein might be adapted to deliver transgenes for treating a variety of diseases, such as, for example, Parkinson’s disease, Alzheimer’s disease, Amyotrophic lateral sclerosis (ALS), Huntington’s disease, Epilepsy, Multiple sclerosis, Spinal muscular atrophy (SMA), Glioblastoma, Hemophilia A, Hemophilia B, Wilson’s disease, Alpha-1 antitrypsin deficiency, Chronic hepatitis B, Chronic hepatitis D, Ornithine transcarbamylase deficiency, Severe Combined Immunodeficiency (SCID), Beta-thalassemia, Sickle cell disease, Graft-versus-host disease (GvHD), Lupus, Rheumatoid arthritis, Lymphoma, Leukemia, Hepatocellular carcinoma, Brain tumors, Metastatic solid tumors, Cystic fibrosis, Pulmonary alveolar proteinosis, Surfactant protein deficiency, Duchenne muscular dystrophy (DMD), Familial hypertrophic cardiomyopathy, Dilated cardiomyopathy, Pompe disease, Peripheral artery disease, and the like.BRIEF DESCRIPTION OF THE DRAWINGS

[0076] FIG. 1 illustrates an FIV-based cargo strand and a modified FIV-based construct configured for targeted genomic integration and inducible expression of therapeutic genes.

[0077] FIG. 2 illustrates the organization of the feline immunodeficiency virus (FIV) genome.

[0078] FIG. 3 provides a schematic overview of the evolution of lentiviral vector systems from first- to third-generation configurations.

[0079] FIG. 4 provides a schematic depiction of fourth-generation lentiviral vector systems.

[0080] FIG. 5 presents a schematic depiction of the genetic mechanism of transduction and regulation conferred by the FIV-based lentiviral vector system described herein.DETAILED DESCRIPTION

[0081] In various embodiments as described herein, the eradication of infectious HIV can be achieved by using an inducible, replication-competent, conditionally auto-inhibiting vector. The inventor recognized, that an inducible, replication-competent, conditionally autoinhibiting lentiviral solves the problem of delivering anti-HIV genetic strategies in vivo, safely and on a long-term basis, after a single administration.

[0082] In various embodiments, vectors, method, and kits provided herein provide an approach that safely and effectively eradicates HIV from an infected person, without the useInventor: Laura A. Prendergast of exogenously administered pharmaceutical cocktails that have been standard practice in the field.

[0083] Provided herein is a gene delivery vector for delivering a dual-function protein for eradicating HIV. The inventor has discovered that combining two functional domains in a dual-function protein can link the mechanism of trapping and eliminating HIV virions to the mechanism of auto-inhibition of the delivery vector’s replication to provide a therapy for eradicating infectious HIV that is auto-inhibiting, conditioned on the elimination of the target HIV virus.

[0084] In some embodiments, described herein is an eradicating cure for the HIV virus. The term “eradicating” is specific, and well known to persons of skill in the art. An eradicating cure for HIV is defined as the complete and permanent elimination of all replication- competent HIV from the body, including from latent reservoirs, without the need for ongoing antiretroviral therapy (ART).

[0085] In various embodiments, the lentiviral vectors, or delivery vectors, and methods described herein provide a functional cure for HIV. As described herein, a “functional” cure for HIV is defined as a durable state of viral remission in which the individual maintains undetectable or extremely low levels of HIV without antiretroviral therapy (ART), with no disease progression and minimal or no risk of viral transmission.

[0086] Provided herein in various embodiments, is a lentiviral-based gene delivery vector capable of delivering the dual-function protein to a patient, or person, with HIV that is in need of treatment. In various embodiments, this dual function is accomplished by 2STOP, a dual-function protein comprised of a transcriptional repressor domain for auto-inhibition of the vector, which is fused to a domain with dominant negative function that inhibits HIV virions from budding out of the membrane of HIV-infected cells.

[0087] The terms "patient" or "individual" or "subject" are used interchangeably herein, and refer to an animal in need of treatment or to be treated. In various embodiments, the animal is a mammal. In some embodiments, the mammal is a primate. In some embodiments, the primate is a human.

[0088] As used herein, a "host cell" is a cell that integrates a heterologous nucleic acid insert of the present disclosure into its genome after introduction of the transgenes by means of the lentiviral vector described herein.

[0089] In some embodiments, the vectors described herein and methods of administering the vectors, can find use in experimental animals and in veterinary applications.Inventor: Laura A. Prendergast

[0090] “FIV lentiviral vector construct”, “FIV vector,” and “recombinant FIV vector” refers to a nucleic acid construct which carries, and within certain embodiments of the invention, is capable of directing the expression of a sequence(s) or gene(s) of interest, and which are packaged into a feline immunodeficiency virus coat for in vivo transduction of cells in a person in need of treatment.

[0091] As used herein, the terms “lentiviral vector”, “lentivector,” “lentiviral gene-delivery vector,” and “lentiviral-based gene-delivery vector” are equivalent and used interchangeably.

[0092] As used herein, a “lentiviral vector” is a delivery system for introducing therapeutic transgenes into a subject, wherein the delivery vector is a lentivirus. A lentiviral vector, as used herein in various embodiments, is a vector that makes use of a lentivirus shell to deliver the transgenes. In some embodiments, the delivery vector is the feline immunodeficiency virus (FIV).

[0093] “Transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been synthesized or isolated from one organism and is introduced into a different organism. This non-native segment of DNA may retain the ability to produce RNA or protein in the transgenic organism, or it may alter the normal function of the transgenic organism's genetic code.

[0094] In some embodiments, the feline immunodeficiency virus’s replication strategy makes use of reverse transcriptase to convert its RNA genome into a double-stranded DNA intermediate, which is then stably integrated into the host genome via viral integrase. As used herein, “reverse transcriptase” refers to an RNA-dependent DNA polymerase enzyme that catalyzes the synthesis of complementary DNA (cDNA) from an RNA template.

[0095] In some embodiments, the feline immunodeficiency virus’s replication strategy makes use of integrase to catalyze the insertion of a reverse-transcribed DNA copy of the viral genome into the host cell’s chromosomal DNA, thereby enabling stable integration and longterm expression of viral or therapeutic genetic material. As used herein, the term “integrase” refers to a viral-encoded enzyme that facilitates the integration of a reverse-transcribed DNA copy of a viral or vector genome into the host cell’s chromosomal DNA. Integrase catalyzes the processing of viral DNA ends and their insertion into the host genome, a critical step in the life cycle of retroviruses and lentiviral vectors.

[0096] As used herein, the term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either singlestranded or double stranded.Inventor: Laura A. Prendergast

[0097] Vector constructs provided herein, in various embodiments, comprise nucleic acids encoding a promoter operably linked to a transgene. The term “nucleic acid” will generally refer to at least one molecule or strand of DNA, RNA or a derivative or mimic thereof, comprising at least one nucleobase, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T,” and cytosine “C”) or RNA e.g., “A,” “G,” uracil “U,” and “C”). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide.” The term “oligonucleotide” refers to at least one molecule of between about 3 and about 100 nucleobases in length. The term “polynucleotide” refers to at least one molecule of greater than about 100 nucleobases in length.

[0098] A “heterologous nucleic acid insert”, as used herein, refers to a nucleic acid sequence to be inserted into a host cell genome that is not derived from the same species or cell type as the host cell.

[0099] As used herein, “nucleic acid packageable size” refers to the total length (in bases) of nucleic acids that will be packaged into a lentiviral vector capsule. In some embodiments, the nucleic acid packageable size is 8,000 - 9,500 bases.

[0100] Terminal repeats of the present disclosure flank minimal intervening viral sequences. As used herein, “minimal intervening viral sequences” are the shortest sequences derived from virus that allow the integration, replication, and packaging of the packageable vector RNA in a host cell.

[0101] In the present disclosure, in various embodiments, described herein is a therapeutic transgene designated 2STOP encoding a dominant-negative domain designated DN-lirl, which traps the HIV virus and prevents mature virions from escaping infected cells. As used herein, a “dominant-negative” portion of an expressed protein refers to a domain that interacts with the same targets as the wild-type gene product, but interferes with the normal function of the wild-type product, thereby reducing its activity. Dominant negative mutations are characterized by a dominant or semi -dominant phenotype, and usually result in loss of function.

[0102] In various embodiments, the 2STOP gene encodes a transcriptional repressor domain to mediate auto-inhibition of the lentiviral vector. In various embodiments, the transcriptional repressor comprises a nuclear localization signal to direct the 2STOP gene product to the nucleus. As used herein a “nuclear localization sequence” (NLS) is a sequence that promotesInventor: Laura A. Prendergast the translocation of a gene product containing the NLS to the nucleus of a host cell to accomplish auto-inhibition of the vector’s replication mechanism.

[0103] As used herein the term “packaging signal” refers to a region of RNA in the FIV genome that is important for efficient RNA packaging during the assembly of FIV into virions. As used herein, “packageable vector RNA” refers to RNA encoding any genetic element, such as a virus, virion, capsid, etc., that is capable of replication when associated with the proper control elements, and can be packaged into an appropriate capsule for delivery between and into cells.

[0104] As used herein, “packaging sequences” are nucleic acid (e.g., RNA) sequences that promote packaging of a viral genome into a capsid. In some embodiments, packaging sequences of the nucleic acids comprise a sequence and the Rev-Responsive Element (RRE). In some embodiments, the sequence comprises the R / U3 portion of the FIV 5’ LTR, and the first 311 bps of the gag coding sequence.

[0105] As used herein, the “nucleocapsid protein packaging target site ( )” is a nucleic acid motif involved in regulating the packaging of a viral genome (e.g., packageable vector RNA) into a capsid. The nucleocapsid protein packaging target site, also referred to as packaging sequences, form secondary structures (e.g., stem-loop, bulges) that are recognized and bound by viral packaging proteins.

[0106] As used herein, “capsid” is the three-dimensional protein shell that encapsulates the genetic material (e.g., packageable vector RNA) of a virus. The capsid may be comprised of heterologous envelope proteins for pseudo-typing the lentiviral vector, to expand the range of host cells that might be transduced.

[0107] As used herein, “pseudo-typing” refers to the inclusion of foreign viral envelope proteins in the lentivector envelope to expand the cellular targets of the lentivector.

[0108] As used herein, “transcribed nucleic acids” refers to nucleic acids that are transcribed in a cell.

[0109] The terms "sequence identity" or "identity" associated with a nucleic acid or polypeptide sequence refer to a nucleic acid base or amino acid in two sequences that are identical when aligned for maximum matching across a particular comparison window.

[0110] The term "sequence identity percentage" refers to a value determined by comparing two properly aligned sequences throughout the comparison window. The portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to reference sequences (without additions orInventor: Laura A. Prendergast deletions) for optimal alignment of the two sequences. This percentage determines the number of positions where the same nucleobase or amino acid residue occurs in both sequences. The percentage is obtained by dividing the number of matching positions by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage. Percent identities can be determined by a person of skill in the art using known analytical approaches and sequence analysis tools such as those, for example, summarized in Washietl, S., & Hofacker, I. L. (n.d.). Chapter 1 Nucleic Acid Sequence and Structure Databases. 3-15.

[0111] The vector described herein, in various embodiments, solves the problem of safe, long-lasting gene delivery and averts the problem of insertional mutagenesis by introducing its genetic cargo site-specifically into safe harbor loci in the genome of the recipient’s cells. As used herein, “insertional mutagenesis” refers to the creation of mutations in DNA by the addition of one or more base pairs, such as might happen when a vector introduces transgenes into the genome of the recipient.

[0112] The vector described herein, in various embodiments, averts the problem of oncogenic transformation or other positional effects by introducing its genetic cargo site- specifically into safe harbor loci in the genome of the recipient’s cells. As used herein, "oncogenesis,” or “oncogenic transformation” also known as "carcinogenesis,” is the formation of a cancer, whereby normal cells are transformed into cancer cells. This comes about as a consequence of mutations in DNA that disrupts normal cell processes such as proliferation and programmed cell death, leading to uncontrolled cell division and formation of tumors in the body.

[0113] The vector described herein, in various embodiments, solves the problem of safe, long-lasting gene delivery and averts the problem of positional effects by introducing its genetic cargo site-specifically into safe harbor loci in the genome of the recipient’s cells. As used herein, the term "positional effects" refers to the influence that the genomic location of an integrated nucleic acid sequence exerts on its transcriptional activity. Such effects may arise due to the local chromatin structure, proximity to endogenous regulatory elements (e.g., enhancers or silencers), or transcriptional interference from neighboring genes. Positional effects can result in variable or unpredictable expression levels of the integrated transgene.

[0114] As used herein, a “safe harbor locus” (also known as a “safe harbor site”, or SHS) is defined as a chromosomal location where therapeutic transgenes can be introduced and whereInventor: Laura A. Prendergast the transgenes can perform their intended function without disrupting endogenous gene activity or causing neoplastic transformations or other positional effects.

[0115] As used herein, a "construct" is an artificially generated segment of nucleic acid that is transferred or cloned into a target subject, tissue, or a cell. As used herein with respect to nucleic acids, the term "cloning" means: recombinantly produced by genetic engineering using recombinant DNA techniques well known to persons of skill in the art.

[0116] As used herein, the term “synthetic vectors” refers to vectors that are based on polymers or lipids, which on interaction with DNA form polymer / DNA complexes called polyplexes and lipid / DNA complexes called lipoplexes. A range of synthetic and natural polymers has been incorporated into vectors for DNA delivery. Synthetic polymers differ in their chemical composition, number of repeating units, and degree of branching. A number of peptides are also utilized as delivery agents.

[0117] Although synthetic vectors, such as polymer-DNA or lipid-DNA complex have been employed for gene delivery, they lack efficiency for clinical applications. Naturally occurring viruses have evolved to deliver their genetic payload to specific cells. Standard molecular biology techniques are used to swap therapeutic transgenes in place of some or all of the viruses’ original genes. Viral-based vectors can be prepared by methods familiar to persons with ordinary skill in the art, or obtained from biotechnology vendors that engineer vectors for introducing transgenes. Vendors that engineer vectors include, for example, Avid Bioservices, Catalent, Creative Biogene, Creative Biolabs, Voyager Therapeutics, Genethon, Novartis, Vascular Biogenics, Akamis Bio, and the like.

[0118] In some embodiments, the nucleic acid constructs of the present disclosure are cloned using techniques of genetic engineering well known to persons of skill in the art. For example, retroviruses, adenoviruses, herpes simplex viruses and adeno-associated viruses have been engineered for delivering transgenes to treat severe combined immuno-deficiency, cystic fibrosis, hemophilia A, Alzheimer’s disease, various cancers and the like.

[0119] The term “protective transgene” as used herein refers to a gene or genetic material containing a gene sequence that has been synthesized or isolated from one organism and introduced into a different organism, for the purpose of conferring genetic or cellular defenses against viral infection, genetic pathologies or other diseases. 2STOP can be described as a protective transgene.

[0120] As used herein, the term “adjunctive protective transgene” refers to an additional gene or genetic material besides the 2STOP, for the expression of a gene product that isInventor: Laura A. Prendergast synthesized or isolated from one organism for the purpose of conferring genetic or cellular defenses against viral infection, genetic pathologies or other diseases. In various embodiments, the adjunctive protective transgenes used to target HIV are nucleic acid aptamers or protein aptamers, immunologic agents, shRNA, and CRISPR-based gene-editing technology targeting HIV sequences for knockout of HIV gene products or excision of HIV sequences from the genome.

[0121] As used herein, “nucleic acid aptamers” refer to small single-stranded RNA or DNA molecules, 20-80 nucleotides in length, that form a tertiary structure enabling them to bind to specific targets. Aptamers can modulate cell signaling or target ligands cell- or tissue-specifically. As used herein, “protein aptamers” refer to short, structured peptide or protein domains engineered to fold into stable conformations that bind specific molecular targets with high affinity and specificity, analogous in function to nucleic acid aptamers.

[0122] As used herein, “immunologic agents” are antibodies, or fragments thereof, targeting the HIV virion coat or HIV gene products. In various embodiments, immunologic agents are selected from a group consisting of monoclonal antibodies (mAbs), broadly neutralizing monoclonal antibodies (bNAbs), monovalent antibodies (MAbs), antigen-binding fragments (Fabs), anti -idiotypic antibodies, camelid-derived nanobodies, other antibody -derived constructs, and functional equivalents thereof.

[0123] As used herein, the term “monoclonal antibodies” refers to antibodies that are produced by a single clone of B cells, and are therefore identical in structure and specific for a single epitope.

[0124] As used herein, the term “broadly neutralizing antibodies” refers to a subset of antibodies that can recognize and neutralize a wide range of viral strains, often across multiple clades or subtypes of a given virus.

[0125] As used herein, the term “monovalent antibodies (MAbs)” refers to antibodies that have only one antigen-binding site. This means it can bind to only one epitope (specific part of an antigen) at a time. This is in contrast to typical antibodies which are bivalent and can bridge two identical antigens, enabling clustering, receptor aggregation, or immune activation.

[0126] As used herein, the term “antigen-binding fragment (Fabs)” refers to the region on an antibody that binds antigens. The Fab comprises one constant and one variable domain from both the heavy and the light chain of the original antibody. The variable domains together comprise the antigen-binding site at the amino terminal end of the monomer.Inventor: Laura A. Prendergast

[0127] As used herein, the term “anti -idiotypic antibody” refers to an antibody that binds to the idiotype of another antibody. An idiotype is a specific region based upon the variable region (Fv) within an antibody which binds to the antigen specifically recognized by that antibody. When one antibody binds to an idiotype of another antibody it is referred to as an anti -idiotypic antibody.

[0128] As used herein, the term “camelid nanobodies” refers to single-domain antibody fragments derived from the heavy-chain-only antibodies found naturally in camelids (e.g., camels, llamas, and alpacas). These nanobodies consist of just the variable domain of the heavy chain (VHH), yet retain full antigen-binding capacity.

[0129] As used herein, the term “gene-editing” refers to alteration of the genetic material of a living organism by inserting, replacing, or deleting a DNA sequence by means of gene editing technology. Gene editing technology is accomplished using zinc finger nucleases (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspersed short palindromic repeats (CRISPR)-associated endonuclease (Cas).

[0130] The terms “functional equivalent” or “functionally equivalent fragment” as used herein, means a protein sequence or nucleic acid sequence that maintains sufficient functionality to be useful in various embodiments described herein.

[0131] The terms “functional fragment”, and “functionally equivalent fragment” of Cas endonuclease are used interchangeably herein to indicate CRISPR-associated endonucleases (Cas enzymes) that recognize and cleave a specific target site in the subject’s genome, guided by signal guide RNA (sgRNA) sequences.

[0132] The terms “functional fragment”, and “functionally equivalent fragment” optionally refer to a portion or subsequence of a Cas endonuclease sequence that retains the ability to perform nicking or cleavage (including single-strand or double-strand breaks).

[0133] The terms “functional variant”, and “functionally equivalent variant” of Cas endonuclease are used interchangeably herein to indicate Cas endonuclease that can recognize a target site. This includes variants of Cas endonuclease that retain the ability to bind, and optionally nick or cleave (insert single-strand or double-strand breaks). Fragments and variants of Cas endonuclease can be obtained by site-directed mutagenesis and synthetic construction.

[0134] As used herein, a “regulatory sequence” is defined as a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of specific genes withinInventor: Laura A. Prendergast an organism. In the present disclosure, in various embodiments, regulatory sequences refer to operator sequences or promotor regions for transcriptional control of coding sequences.

[0135] As used herein, an “operator sequence” is defined as a genetic sequence which is recognized and bound by proteins responsible for mediating transcription. In various embodiments, the operator sequence is selected from a group of sequences recognized and bound by transcriptional repressors.

[0136] As used herein, a “transcriptional repressor” is a DNA-binding protein that binds to its cognate operator and sterically represses the expression of one or more downstream genes. Transcriptional repressors are generally understood by persons of skill in the art to be proteins that bind promoter sequences or operator sequences to impede binding of RNA polymerase although repressors utilizing other mechanisms do exist. Some repressors allow RNA polymerase binding to the promoter, but prevent opening up of the DNA helix at the nucleic acid sequence to be transcribed. Another group of repressors allow opening of the DNA helix, but prevent RNA polymerase from transcribing the downstream sequences.

[0137] In various embodiments, the operator sequences recognized by the transcriptional repressor domain of the 2STOP have “dyad symmetry,” which refers to two areas of a DNA strand whose base-pair sequences are inverted repeats of each other.

[0138] A DNA-binding repressor blocks the attachment of RNA polymerase to the promoter, thus inhibiting transcription of downstream genes into messenger RNA. In various embodiments the transcriptional repressors are selected from a group that includes, for example, Gal4-BD, GalR, GalS, LacI, Cep A, CytR, Mall, PurR, RafR, RbtR, ScrR, and functional equivalents.

[0139] In various embodiments, the DNA-binding transcriptional repressor domain of the 2STOP comprises a nuclear localization sequence. The term “nuclear localization sequence” (NLS) is known to persons with skill in the art, and refers to a domain in a protein that allows translocation of the molecule containing the NLS into the nucleus of the host cell receiving the transgenes.

[0140] As used herein, the term “cognate operator sequence” refers to a nuclear regulatory sequence that is specifically recognized and bound by a specific transcriptional repressor.

[0141] As used herein, the term “cognate transcriptional repressor” refers to a transcriptional repressor that specifically recognizes the specific operator sequence downstream of the 5 ’ LTR.Inventor: Laura A. Prendergast

[0142] As used herein, a “promoter” refers to a regulatory DNA sequence, that is required to initiate the transcription of a gene.

[0143] As used herein, a “promoter” is a sequence of DNA to which proteins bind to initiate transcription of a gene. In various embodiments, the promoter sequence may be a constitutive promoter. In various embodiments, the promoter sequence may be a cell-type specific promoter. Promoters that may be utilized are known in the art and can be heterologous, native, inducible, constitutive, and / or tissue specific. Promoters described herein, in various embodiments, are operably linked to transgenes.

[0144] As used herein, “operably linked” sequences include expression control sequences that can be contiguous with, or at some distance from the gene of interest. For example, a promoter region is operably linked to a nucleic acid sequence if the promoter region can affect transcription of that nucleic acid sequence such that the resulting transcript can be translated into the desired protein or polypeptide. The phrases “operatively positioned,” “under control,” or “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the transgene to control RNA polymerase initiation of an operably linked coding sequence.

[0145] As used herein, “heterologous promoter” indicates a regulatory sequence experimentally introduced into a cell that does not normally contain that sequence. Heterologous refers to the fact that the introduced genetic material has been cloned or derived from a different cell type or species from the recipient of the heterologous genetic material. Examples of heterologous promoters that can be used in various embodiments described herein include, for example, promoters that are native to FIV such as the 5’ Long Terminal Repeat (LTR) and the 3’ LTR for replication of vector sequences. In various embodiments, heterologous promoters are used to drive expression of the 2STOP gene.

[0146] As used herein, a “constitutive promoter” is defined as a segment of nucleic acid that functions as a promoter that allows continuous transcription of an operably linked coding sequence. In various embodiments, the constitutive promoter is selected from a group which includes hCMV, EF-1 alpha, CAGG and SV40 and functional equivalents thereof.

[0147] As used herein, the term “constitutive internal promoter ” refers to a promoter sequence located within a gene expression construct that drives continuous, unregulated transcription of an operably linked transgene, independent of cell type or external stimuli. In certain embodiments, the promoter is positioned downstream of the 5' LTR and functions independently of LTR-driven transcription. Exemplary constitutive internal promotersT1Inventor: Laura A. Prendergast include, without limitation, the hCMV, EFla, SV40, and CAG promoters, as well as synthetic or derivative variants thereof.

[0148] As used herein, a “cell-type specific promoter” is defined as a segment of nucleic acid that functions as a promoter that allows transcription of a downstream coding sequence, and which is transcriptionally active only in specific cell types. In various embodiments, the cell-type specific promoter is selected from a group that includes CD4, CD8, and CD34 and functional equivalents.

[0149] As used herein, the term “human optimized” refers to genetic or amino acid sequences that have been constructed using codons that are preferentially utilized in humans. Because the genetic code is “degenerate,” the same amino acid may be encoded by multiple codons. It has been found that different organisms exhibit preferential usage of certain codons over the others. It has also been discovered that codon usage bias will significantly affect protein expression. When a desired transgene is conferred on an organism not normally expressing that gene, it is useful to optimize the codons for the host organism. Codon optimization may improve DNA cloning efficiency, improve RNA stability, and improve transcription or translation efficiency.

[0150] Fragments or functional equivalents of sequences are included in various embodiments, regardless of the degree of homology that they show to the sequences disclosed herein, provided that they maintain functionality, since sequences disclosed herein can be readily modified without any significant effect on the functional activity of the resulting fragment.

[0151] As used herein, a “therapeutic gene” is a nucleic acid sequence (i.e. “transgene”) that encodes a protein with an anti-HIV function. Transgenes that may be introduced, in various embodiments described herein, include, for example, 2STOPA, 2STOPB, mAbs, MAbs, bNmAbs, Fabs, nucleic acid aptamers, protein aptamers, shRNA that can be processed into RNAi for knockdown of HIV gene products, CRISPR-associated proteins (Cas endonucleases) with signal guide RNA (gRNA) sequences for site-specific insertion of lentivector constructs, anti -idiotypic antibodies, and the like.

[0152] In some embodiments, constructs as described herein comprise more than one promoter (e.g., 2, 3, 4, 5, or more promoters). In some embodiments, one or more of the promoters in a construct described herein is an “internal promoter”. As used herein, an internal promoter refers to a promoter that is operably linked to expression of the therapeuticInventor: Laura A. Prendergast transgene within the packageable vector RNA but separate from the endogenous lentiviral promoter that drives replication of the entire vector construct.

[0153] In various embodiments described herein, the lentiviral vector also comprises auxiliary transgenes for targeting circulating anti-HIV antibodies. As used herein, the term “auxiliary transgene” refers to a genetic sequence or sequences that are delivered by means of the lentiviral vector to accomplish seroreconversion. As used herein, “seroreconversion” is a return to the HIV-negative serotype.

[0154] As used herein, “effective dose” is defined as a dose of vector that delivers sufficient levels of the therapeutic genes to the recipient to eradicate the HIV virus from the cells and tissues of the patient. An effective dose can be calculated by a person of skill in the art, based on the desired multiplicity of infection, and a cell count of the patient’s T-cells. See, for example, Saenz et al. Feline immunodeficiency virus-based lentiviral vectors. Cold Spring Harb Protoc. 1, 71-76 (2012).

[0155] As used herein, the term “multiplicity of infection (MOI)” refers to the ratio of transducing agents (i.e., lentivector) to infection targets (i.e., recipient’s cells). For example, when referring to a group of cells inoculated with virus particles, the MOI is the ratio of the number of virus particles to the number of target cells present in a defined space.

[0156] The FIV lentivector particles is prepared for delivery into a subject in need of treatment suspension in a sufficient amount of a formulation buffer, which is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water. As used herein, a “buffering compound” or “buffering component” should be understood to refer to a substance that functions to maintain the aqueous suspension at a desired pH.

[0157] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.MOLECULAR BIOLOGY OF FIV-BASED LENTIVIRAL VECTORS

[0158] In recent years, numerous viruses with features that hold promise for in vivo gene delivery have been identified. Recombinant viruses such as adenoviruses, adeno-associated viruses, herpes viruses, poxviruses, retroviruses and more recently lentiviruses, are increasingly being used in the laboratory and clinic.

[0159] Vectors derived from lentiviruses provide effective gene delivery systems. The ability of lentivector s to transduce non-dividing cells makes them useful for gene delivery toInventor: Laura A. Prendergast post-mitotic, highly differentiated cells. Human gene therapy clinical trials are currently under way using lentivectors in a wide range of human diseases.

[0160] Lenti viruses are a sub-category of Retroviruses. Retroviridae are so named because their genome is comprised of RNA, which is copied into DNA by means of reverse transcriptase when they infect a cell. Lentiviruses are so named because they cause diseases with long incubation periods; they develop “slowly.” Described herein in various embodiments is a lentiviral vector encoding and capable of delivering a therapeutic gene in vivo to eradicate human immunodeficiency virus (HIV) from infected cells in infected humans.

[0161] The morphology and genome across the lentivirus genera are essentially similar. Lentiviruses are roughly spherical particles with a diameter of approximately lOOnm. The diploid genome of the feline immunodeficiency virus (FIV) consists of two single-stranded (+) RNA molecules. The FIV viral genome is packaged by nucleocapsid proteins and bound to an integrase, reverse transcriptase, and a protease forming a cone-shaped isometric core. In some embodiments, the FIV viral genome is packaged by a nucleocapsid protein set forth in SEQ ID NO: 322, and bound to an integrase set forth in SEQ ID NO: 317, a reverse transcriptase set forth in SEQ ID NO: 316, and a protease set forth in SEQ ID NO: 318, forming a cone-shaped isometric core. The core is encased in a shell of capsid proteins, which in some embodiments are set forth in SEQ ID NO: 321, which is in turn surrounded by matrix proteins, in some embodiments set forth in SEQ ID NO: 320, and a lipid envelope. The viral envelope incorporates regularly spaced transmembrane and surface glycoproteins that projects out by about 8nm, creating a rough outer appearance.USE OF FELINE IMMUNODEFICIENCY VIRUS AS A LENTIVIRAL VECTOR

[0162] FIV has been utilized in numerous applications to induce long-term expression of transgenes in vitro. Lentiviral vectors derived from FIV enable efficient in vivo and ex vivo delivery, integration and stable expression of transgenes into dividing, as well as nondividing cells.

[0163] The vector’s genetic cargo is packaged in a feline immunodeficiency virus (FIV) coat. Non-primate lentiviral vectors such as FIV are an attractive option for in vivo gene transfer into humans for several reasons.

[0164] First, unlike other viral-based vectors (such as Adenovirus or Adeno-Associated Virus (AAV), FIV fails to cause any sort of disease in humans. Despite an extensive record of human exposure to wild-type FIV, no human infection or disease has eventuated; evenInventor: Laura A. Prendergast despite the ability of wild-type FIV to gain entry into human cells via a human chemokine receptor.

[0165] Second, FIV fails to cross-react immunologically with HIV. This is desirable so that the gene delivery system is not attacked by the host’s antibodies against the targeted virus. It is not advantageous for the host’s immune system to target the vector that is delivering therapeutic transgenes.

[0166] Third, FIV is capable of transducing both non-dividing and terminally differentiated cells, including hepatocytes, neurons, and myocytes. This characteristic provides a significant advantage over other viral vectors that are unable to cross the intact nuclear membrane, thereby enabling the transduction of a broader range of target cells within the patient.

[0167] Fourth, lentiviral cDNA integrates into the genome of the host cell by means of the viral-encoded integrase enzyme. This ensures that the integrated genes will persist indefinitely throughout the lifetime of the transduced cell, and will be transmitted to daughter cells following mitosis.DESCRIPTION OF FELINE IMMUNODEFICIENCY VIRUS GENOME

[0168] FIG. 2 illustrates the organization of the feline immunodeficiency virus (FIV) genome in various embodiments. Genes and long terminal repeats (LTRs) are shown approximately to scale, reflecting their relative arrangement along the viral RNA. The pol open reading frame is translated as a fusion Gag-Pol polyprotein via a programmed ribosomal frameshift from the full-length genomic RNA. Core structural proteins include MA (matrix), CA (capsid), and NC (nucleocapsid), while enzymatic proteins include PR (protease), RT (reverse transcriptase), DU (dUTPase), and IN (integrase). Several cis-acting RNA elements are annotated above the genome schematic, including: y (packaging signal), required for selective encapsidation of viral RNA into virions; mSD (major splice donor site), essential for generating sub-genomic transcripts; DIS (dimerization initiation site), which mediates RNA- RNA interactions required for genome dimer formation; PBS (primer binding site), where host tRNA anneals to initiate reverse transcription; and RRE (Rev response element), a structured RNA domain that binds the viral Rev protein to mediate nuclear export of unspliced and partially spliced transcripts. Regulatory elements within the LTRs are shown below the main genome map in magnified view. These LTRs contain promoter and enhancer elements that initiate transcription of the integrated provirus and may include binding sites for host transcription factors and viral regulatory proteins.Inventor: Laura A. Prendergast

[0169] The FIV pol sequence encodes the reverse transcriptase (RT) SEQ ID NO: 298, integrase (IN) SEQ ID NO: 299, and protease (PR) SEQ ID NO: 300 enzymes.

[0170] The FIV gag sequence SEQ ID NO: 290 encodes all the gene products necessary for virion assembly and budding. The FIV gag polyprotein SEQ ID NO: 319 is processed by the viral protease into the structural proteins giving shape to the FIV virion including: matrix (MA) SEQ ID NO: 320, capsid (CA) SEQ ID NO: 321, and nucleocapsid (NC) SEQ ID NO: 322.

[0171] The FIV env sequence codes for the envelope glycoprotein SEQ ID NO: 305, which mediates virion attachment and entry into target cells.

[0172] FIV vectors can, in addition, comprise one or more RNA export elements (also variously referred to as RNA transport, nuclear transport or nuclear export elements) which, within one aspect of various embodiments of the lentiviral vector described herein, is the FIV Rev-responsive element (RRE) SEQ ID NO: 293.

[0173] The FIV genome carries an RNA packaging signal ( ) SEQ ID NO: 91 to enclose the genome into the virion capsule. The genome also includes the central polypurine tract (cPPT) SEQ ID NO: 294, required for reverse transcription, and the two long-terminal repeats (LTRs) which function as transcriptional promoter (5’ LTR) SEQ ID NO: 88 or terminator of transcription (3’ LTR) SEQ ID NO: 89, and also serve to integrate the proviral DNA.

[0174] A small set of accessory genes, important for viral replication, immunological response and pathogenesis is also contained in the FIV genome. The rev gene product SEQ ID NO: 308 is essential for viral replication, stabilizing incompletely spliced viral RNAs and transporting viral RNAs from the nucleus to cytoplasm; vif SEQ ID NO: 309 affects the infectivity of the virus; ORF A SEQ ID NO: 310 is not essential but affects the efficient replication of the virus in primary peripheral blood lymphocytes.DESCRIPTION OF FELINE IMMUNODEFICIENCY VIRUS TRANSDUCTION IN HUMANS

[0175] FIV enters into target cells through the interaction of the surface (SU) subunit SEQ ID NO: 306 of the viral envelope glycoprotein (env) SEQ ID NO: 305 with host T-cell receptors CD 134 and CXCR4. The viral and host membranes then fuse, in a process mediated by the transmembrane (TM) subunit SEQ ID NO: 307 of Env. After entry and uncoating, the FIV RNA genome is reverse transcribed into DNA, through the activity of reverse transcriptase (RT) SEQ ID NO: 298, which is delivered by the FIV virus. A host cell tRNALysInventor: Laura A. Prendergast molecule primes reverse transcription of both copies of FIV’s RNA genome. While the FIV genome is being reverse-transcribed, the viral core particle reorganizes, shedding capsid molecules (CA) SEQ ID NO: 303. The partly disassembled core particle translocates to the cell nucleus with the viral genome as the pre-integration complex (PIC), and the viral cDNA is integrated into the host genome by the action of integrase (IN) SEQ ID NO: 299. The provirus persists in the host cell genome throughout the life of the cell and is passed on to daughter cells after cell division.

[0176] After integration, RNA polymerase II transcribes full-length genomic RNA from the proviral genes. Transactivation of the FIV genome is accomplished by means of the ORF A accessory gene product SEQ ID NO: 310, which activates transcription from the FIV 5’ LTR. Virus particles are packaged and released from the infected cell, budding out of the host cell, along with viral envelope glycoproteins previously incorporated into the host cell membrane.

[0177] Expression of lentiviral transcripts is controlled qualitatively and quantitatively through multiple splice sites. The Regulator of viral expression (Rev) protein SEQ ID NO: 308 works in conjunction with the host’s nuclear export protein (CRM-1) and the FIV- encoded Rev -response element (RRE) SEQ ID NO: 293, a structured feature of the RNA genome, to export viral transcripts out of the nucleus. FIV’s Rev protein SEQ ID NO: 308 is expressed in the host cell, enabling transport and expression of spliced and un-spliced viral RNAs.

[0178] Expressed gag protein SEQ ID NO: 301 perform diverse functions which include packaging of the viral genomic RNA. The first 311 bp of the gag sequence SEQ ID NO: 290 serve as a packaging signal, ( ) SEQ ID NO: 91, which, in conjunction with the 5’LTR SEQ ID NO: 88, is sufficient for encapsidation of genomic RNA. The capability of gag SEQ ID NO: 301 to identify viral genomic RNA from a large population of other RNAs inside the host cytoplasm is mediated by nucleocapsid (NC) SEQ ID NO: 304, which recognizes the viral packaging signal ( ) SEQ ID NO: 91.FELINE IMMUNODEFICIENCY VIRUS-BASED VECTOR TECHNOLOGY

[0179] Until now, the entire focus of lentiviral -based vector design has been to avoid the production of Replication-competent Lentiviruses (RCLs) by using a “split genome” strategy. The genes encoding elements of the packaging system are delivered as three or more separate plasmids with almost no homology overlap, to minimize the chance of multiple independent recombination events leading to generation of an RCL.Inventor: Laura A. Prendergast

[0180] The original two-plasmid FIV vector system, was engineered using a clone of FIV (see FIG. 2 for the genome of wild-type FIV) and established that FIV vectors could be used to transduce non-dividing human cells. As expression from the endogenous FIV promoter was found to be minimal in transduced human cells, a heterologous promoter - human Cytomegalovirus (hCMV) promoter - was engineered into the FIV genome replacing the 5’ FIV U3, at the TATA box upstream of the Repeat region.

[0181] To improve the safety profile of the original proof-of-concept, this system was extensively re-engineered. Second generation vectors deleted additional virulence genes ( if and or / 2) from the first construct, attenuating FIV in vivo. Third generation vectors moved Regulator of Viral Expression (rev) to a separate construct.

[0182] FIG. 3 illustrates the evolution of lentiviral vector systems from first- to third- generation designs. Panel A shows first-generation vectors using a single packaging plasmid encoding all HIV-1 genes except env, which is provided separately as VSV-G; the transfer plasmid includes a transgene cassette flanked by full-length LTRs. Panel B depicts second- generation vectors with improved safety via deletion of accessory genes (vi vpr, vpu, nef). Panel C shows third-generation vectors requiring four plasmids: gag-pol, rev, envelope, and transfer. Tat-independence is achieved by replacing the 5' LTR U3 region with a heterologous promoter (e.g., CMV), and self-inactivating function is conferred by partial deletion of the U3 region in the 3' LTR. Additional elements such as cPPT / FLAP and WPRE enhance nuclear import and transgene expression. All components are shown schematically and not to scale.

[0183] Fourth generation vectors placed gag and pol onto separate constructs. Third and fourth generation vectors also added self-inactivating (SIN) sequences that reduced expression from the viral LTRs.

[0184] FIG. 4 illustrates a fourth-generation lentiviral vector system comprising three plasmids: a transgene vector with self-inactivating (SIN) LTRs, RRE, cPPT, WPRE, and an internal promoter driving the therapeutic transgene; a packaging vector split into two plasmids encoding codon-optimized gag-pol and rev, each under heterologous promoters and devoid of accessory genes; and an envelope vector encoding VSV-G under CMV control. This modular system enhances biosafety, transcriptional control, and pseudotyping flexibility. ADVANTAGES OF A REPLICATION-COMPETENT LENTIVIRAL VECTOR

[0185] The lentiviral vector, described herein in various embodiments, provides significant advantages over current gene delivery technology. Prior to the inventor’s discovery of aInventor: Laura A. Prendergast replication-competent, auto-inhibiting vector, vectors for viral-based gene delivery strategy had been designed to avoid the development of replication-competent lentivirals (also herein called “replication-competent lentiviral vectors” or “RCLs”) that could potentially recombine with target viruses with deleterious effects for the patient receiving the transgenes. To avert this, early gene delivery vectors were segregated into three or more functional constructs (usually envelope, packaging, and transfer constructs), with the packaging construct modified to prevent viral sequences from being packaged into the vector envelope.

[0186] An advantage presented by an auto-inhibiting vector, in various embodiments described herein, is that it circumvents the problems associated with replication-competent lentivectors (RCLs). When treating HIV, it is desirable that the delivery system offer ongoing protection so that it can transduce new cohorts of cells with the therapeutic genes. Similarly, it is desirable that the therapeutic genes are switched on (“induced”), only when necessary.

[0187] In certain embodiments, the lentiviral vector of the present disclosure is configured for prophylactic administration to subjects who are not infected with HIV, thereby functioning as a vaccine. Upon administration, the vector delivers one or more protective transgenes, optionally in combination with adjunctive protective transgenes, which integrate into the genome of the recipient’s cells. In the absence of HIV infection, expression of the protective transgenes remains in a dormant or repressed state. Upon subsequent exposure to HIV, the vector-encoded protective transgenes are activated, thereby eliciting antiviral activity sufficient to prevent establishment of infection and / or to eradicate HIV from the host.

[0188] In some embodiments, the lentiviral vectors of the present disclosure are engineered to minimize or eliminate the risk of generating replication-competent lentiviral vectors (RCLs) through recombination with infectious HIV. In certain embodiments, the vector backbone is derived from feline immunodeficiency virus (FIV) and is modified to substantially remove any nucleotide sequences that are homologous to, or capable of recombining with, HIV genomic sequences. In some embodiments, any residual sequences that retain limited homology to HIV are human-codon-optimized to further reduce recombination potential. In the unlikely event of recombination between the FIV-based vector and infectious HIV, the integrated therapeutic cargo — including the dual-function 2STOP protein and any adjunctive anti-HIV strategies encoded therein — remains operative to neutralize and eliminate the resulting recombinant virus.

[0189] The lentiviral vector described herein in various embodiments is FIV-based, and therefore has minimal sequence similarities with the HIV genome. The only HIV-derivedInventor: Laura A. Prendergast sequence included in the lentiviral FIV vector is the 59-base Transactivator Responsive Region (TAR) sequence SEQ ID NO: 57 that precedes the 2STOP coding sequence, and 126 bases in the DN-lirl coding sequence SEQ ID NO: 46. Minimal sequence overlap diminishes the risk of the FIV vector recombining with HIV genetics. Moreover, comparison of the nucleotide sequences for the HIV LTRs and the FIV LTRs shows no significant homology, suggesting that it is unlikely the HIV genome will recombine into the FIV genome in such a way as to become replication-competent.MOLECULAR GENETICS OF CONDITIONAL AUTO-INHIBITION: A NESTEDREGULATORY SYSTEM

[0190] The vector described herein bears a unique nested genetic mechanism, enabling it to deliver its transgenes, then auto-inhibit its own replication until the cells bearing therapeutic transgenes are challenged with infectious HIV.

[0191] The lentiviral vector genome, in various embodiments as described herein, comprises the 2STOP coding sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and functional equivalents thereof.

[0192] In some embodiments, the 2STOP coding sequence is preceded by a Transactivator Responsive Region (TAR), SEQ ID NO: 57 which is normally recognized by HIV's transactivator molecule (tat). TAR-tat interaction stabilizes the RNA construct following the TAR region leading to 100-fold upregulation of the gene following the TAR sequence. In various embodiments, the gene following the TAR sequence is the 2STOP.

[0193] Following integration, in some embodiments, expression of the 2STOP gene is driven by a constitutive promoter selected from the group consisting of hCMV, EF-1 alpha, CAGG and SV40 and functional equivalents thereof. In some embodiments, the constitutive promoter sequence is selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84 and functional equivalents thereof.

[0194] In some embodiments, the therapeutic dual-function protein 2STOP comprises a transcriptional repressor domain, such as, for example, the DNA-Binding Domain of the yeast GAL4 region (GAL4-BD) SEQ ID NO: 47, which is fused in-line with a dominantnegative domain. In some embodiments, the dominant-negative portion of the 2STOP,Inventor: Laura A. Prendergast designated DN-lirl SEQ ID NO: 46, traps HIV virions at the cell membrane, preventing them from budding out of the cell.

[0195] In some embodiments, the dominant-negative portion of the 2STOP, designated DN- lirl SEQ ID NO: 46, traps HIV virions at the cell membrane, preventing them from budding out of the cell. Wherein HIV particles trapped by the dominant negative DN-lirl domain SEQ ID NO: 46 of the dual-function 2STOP are routed into endogenous digestive pathways and enzymatically eliminated.

[0196] In some embodiments, the transcriptional repressor domain of the 2STOP is selected from the group consisting of GAL4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR, and other transcriptional repressors known in the art. In some embodiments, the transcriptional repressor comprises a nuclear localization sequence (NLS) for translocation of the 2STOP protein to the nucleus of a subject’s cell. Upon eradication of infectious HIV, the nuclear localization signal contained within the transcriptional repressor domain directs translocation of the remaining 2STOP product to the nucleus.

[0197] In various embodiments, the modified wild-type and cargo FIV constructs comprise an operator sequence with dyad symmetry selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 and functional equivalents thereof, that is placed downstream of the 5’ LTR sequence of the vector construct. In some embodiments, the 5’ LTR sequence is set forth in SEQ ID NO: 88. The transcriptional repressor domain of the 2STOP dual-function protein homo-dimerizes and binds the cognate operator sequence. Binding of the operator by the transcriptional repressor domain of the 2STOP accomplishes steric inhibition of downstream sequences, inhibiting the vector’s replication.

[0198] In various embodiments, upon challenge with infectious HIV, the process of HIV budding out of the membrane recruits the 2STOP protein into the budding HIV virions through interaction of the 2STOP’s dominant negative domain (DN-lirl) domain SEQ ID NO: 46, and away from the operator sequence in the vector’s genome which is bound by the transcriptional repressor portion of the 2STOP.

[0199] In various embodiments, upon challenge with infectious HIV, inhibition of replication of the lentiviral vector described herein is temporarily relieved by recruitment of the transcriptional repressor domain of the 2STOP dual-function protein away from its cognate operator sequence within the vector genome thereby rendering the FIV vectorInventor: Laura A. Prendergast transiently replication-competent. Wherein relief of transcriptional repression of the vector provides for ongoing transduction of new populations of T-cells with the lentivector’s therapeutic transgenes during challenge with HIV.

[0200] In various embodiments, following enzymatic elimination of the trapped HIV virions, the remaining 2STOP protein translocates back to the host cell nucleus by means of the nuclear localization sequence comprised within the transcriptional repressor domain of the 2STOP. In various embodiments, the transcriptional repressor domain of the 2STOP homo-dimerizes, and binds the cognate operator sequence to re-establish inhibition of the lentiviral vector's proliferation to sub-baseline levels.

[0201] The dual-function regulatory protein described herein — modeled on the 2STOP architecture and comprising a dominant negative domain fused to a sequence-specific DNA- binding domain — may be applied broadly across viral -based vectors, not limited to lentiviral systems. Any such vector can be functionally repressed through insertion of an operator sequence downstream of its 5’ LTR or analogous control element, provided the operator is recognized by the cognate repressor domain of the dual-function protein. Upon binding, this protein imposes direct steric hindrance on the transcriptional machinery, thereby suppressing vector replication. The modularity of this system enables its adaptation to diverse viral platforms, offering a generalizable strategy for tunable replication control in gene delivery applications.SITE-SPECIFIC INTEGRATION OF TRANSGENES BY MEANS OF CRISPR- BASED GENE-EDITING

[0202] In certain embodiments, non-integrating lentiviral vectors (NILVs) are employed to reduce the risk of insertional mutagenesis. NILVs comprise a mutated or inactivated integrase gene, preventing stable genomic integration and resulting in episomal DNA containing one or two long terminal repeats (LTRs). Such episomes remain transcriptionally active but, lacking an origin of replication, are diluted during cell division while remaining stable in non-dividing or terminally differentiated cells. Accordingly, NILVs offer a safe and effective platform for gene delivery to non-dividing or differentiated cell populations.

[0203] In certain embodiments, replication-competent gene-delivery systems are designed to overcome limitations of therapies requiring lifelong administration, such as pharmaceutical agents for HIV. For conditions necessitating broad and sustained transduction of diverse cell types, non-integrating vectors offer limited advantage, as repeated dosing remains necessary. An optimal gene therapy would be delivered once, integrate therapeutic sequences into theInventor: Laura A. Prendergast genome of long-lived progenitor cells, such as hematopoietic stem cells (HSCs), and provide durable expression in successive cell generations. However, genomic integration carries inherent risks, including insertional mutagenesis, oncogenic transformation, and other positional effects.

[0204] In certain embodiments, durable protection is achieved by integrating the lentiviral vector cargo into the host genome. Lentiviruses uniquely enable stable genomic integration in both dividing and non-dividing cells, offering an advantage over non-integrating viral vectors such as adenovirus (AV), adeno-associated virus (AAV), alphavirus, herpesvirus, and vaccinia virus, which do not stably integrate their genetic material.

[0205] Site-specific insertion or disruption of genetic material may be accomplished using programmable nuclease technologies, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeat-associated (CRISPR-Cas) systems. ZFNs and TALENs employ engineered DNA-binding domains linked to a nuclease, such as FokI, to induce targeted double-strand DNA breaks. ZFNs utilize zinc finger motifs to bind specific sequences, whereas TALENs, derived from Xanthomonas species, use tandem repeats with variable di-residues that recognize individual base pairs, affording broader targeting capacity. Double-strand breaks generated by these systems are repaired via non-homologous end joining (NHEJ) to disrupt gene function or via homology-directed repair (HDR) to insert donor sequences. Although capable of precise editing, ZFNs and TALENs are generally less favored than CRISPR-Cas due to cost, structural complexity, and reduced targeting efficiency.

[0206] In various embodiments, clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) endonuclease systems are employed for targeted genome modification. Since its initial characterization in 2012, the CRISPR-Cas platform has been widely utilized for precise gene editing. In this approach, a guide RNA (gRNA) directs the Cas endonuclease to a complementary target DNA sequence flanked by a proto-spacer adjacent motif (PAM). Upon binding, the Cas endonuclease cleaves the target DNA, generating a double-stranded break. Subsequent cellular DNA repair processes, including non-homologous end joining (NHEJ) or homology-directed repair (HDR), facilitate targeted disruption of the locus or the insertion of exogenous transgenes.

[0207] In various embodiments, compact CRISPR-Cas orthologues are employed to facilitate efficient packaging within lentiviral vectors for in vivo genome editing. Suitable CasInventor: Laura A. Prendergast orthologues include, without limitation, Staphylococcus aureus-derived saCas9 (3.16 kb) (SEQ ID NO: 92), Campylobacter jejuni-derived cjCas9 (2.95 kb) (SEQ ID NO: 93), Staphylococcus agnetis-derived sagCas9 (2.98 kb) (SEQ ID NO: 94), Cas (SEQ ID NO: 95), and functional equivalents thereof. Upon delivery into a recipient cell, the CRISPR-associated nuclease mediates site-specific integration of the vector genome into the host genome, optionally at a genomic safe-harbor locus, to ensure stable and predictable expression of the therapeutic transgene.

[0208] In some embodiments, the therapeutic lentiviral vector is configured for targeted integration of its cargo into a genomic safe harbor locus of the host cell, thereby minimizing the risk of insertional mutagenesis or other positional effects, and ensuring stable, long-term expression of the therapeutic transgene. Exemplary safe harbor loci include, but are not limited to, the adeno-associated virus integration site 1 (AAVS1, PPP1R12C locus), the CCR5 locus, the ROSA26 locus, the Hippl 1 locus, the HPRT1 locus, and functional equivalents. See e.g., Smith et al., “Lentiviral Vector Integration at the AAVS1 Safe Harbor Site Enables Stable Gene Expression,” Molecular Therapy, Vol. 24, No. 3, pp. 535-546 (2016); Perez et al., “Establishment of HIV- 1 Resistance in CD4+ T Cells by Genome Editing Using Zinc-Finger Nucleases,” Nature Biotechnology, Vol. 26, No. 7, pp. 808-816 (2008);Thompson et al., “Targeted Mutagenesis of the HPRT Gene in Mouse Embryonic Stem Cells,” Cell, Vol. 51, No. 3, pp. 503-512 (1987).

[0209] In various embodiments, sgRNA sequences direct insertion of the FIV constructs into HIV coding sequences for knockout of HIV proteins. In various embodiments, sgRNA sequences direct insertion of the FIV constructs into the human CCR5 receptor to knock out HIV’s primary mode of entry into human cells. In various embodiments, sgRNA sequences direct excision of HIV sequences from the recipient’s genome.

[0210] In some embodiments, CRISPR / Cas genome-editing components are configured to disrupt one or more HIV coding sequences critical for viral replication or infectivity.Target sequences may include the gag-pol region (encoding structural precursors MA, CA, NC, p6, and enzymatic components PR, RT, RNase H, and IN), the env region (encoding envelope glycoproteins gpl20 and gp41), and regulatory genes such as tat and rev. In certain embodiments, CRISPR-based editing may additionally target accessory genes such as, for example, nef vif vpr, and vpu. In further embodiments, sequences derived from HIV-2 or related lentiviruses, such as vpx, can also be targeted for disruption.Inventor: Laura A. Prendergast

[0211] In some embodiments, sgRNA sequences for insertion of the FIV-based vector genome into HIV’s gag-pol sequence are selected from the group consisting of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO:119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO:124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, and the like.

[0212] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s integrase sequences are selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and the like.

[0213] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s env sequence are selected from the group consisting of SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, and the like.

[0214] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vif sequence are selected from the group consisting of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and the like.

[0215] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpu sequence are selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, and the like.

[0216] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpr sequence are selected from the group consisting of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174,SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, and the like.

[0217] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s rev sequence are selected from the group consisting of SEQ ID NO: 179,SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184,SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189,SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the like.Inventor: Laura A. Prendergast

[0218] In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpx sequence are selected from the group consisting of SEQ ID NO. 193, SEQ ID NO. 194, and the like.PREPARATION OF FIV-BASED VECTOR CONSTRUCTS

[0219] Recombinant FIV vector constructs are prepared given the disclosure provided herein and in accordance with methods known to persons of skill in the art.

[0220] In various embodiments, the vector comprises one or more transgenes selected to provide therapeutic benefit against a specific strain or clade of HIV infecting the subject. Selection of such transgenes in various embodiments, is guided by molecular characterization of the subject’s HIV strain, including analysis of viral genome sequence, tropism, resistance profile, and clade classification. This enables the incorporation of strain-matched or cladespecific regulatory, inhibitory, or excision-based elements designed to maximize efficacy and minimize off-target effects. In some embodiments, multiple transgenes may be co-delivered in a combinatorial or modular format to address quasi-species diversity or known resistance mutations.

[0221] In various embodiments, the FIV-based vector comprises a modified wild type FIV strand and an FIV cargo strand.

[0222] In various embodiments, the FIV cargo construct is engineered with the transgenes ordered according to the genome map seen in FIG. 1. Persons of skill in the art can engineer the coding sequences for inclusion in the FIV cargo construct using standard techniques of Molecular Genetics (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manua1,2nded., Cold Spring Harbor Laboratory Press, 1989; Kunkel, PNAS 82:488, 1985).

[0223] In various embodiments, the cargo construct of the lentiviral vector described herein comprises the therapeutic gene, 2STOP. In various embodiments, the 2STOP coding sequence can be selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and functional equivalents thereof. The 2STOP gene product neutralizes HIV and represses the vector’s proliferation. Regulatory genetic sequences of the cargo construct enable the vector’s auto-regulatory mechanism.

[0224] In various embodiments, a constitutive promoter drives expression of 2STOP, to establish baseline inhibition of the vector’s replication in all transduced cells until challengeInventor: Laura A. Prendergast with infectious HIV. In some embodiments, the cargo construct comprises native FIV 5’ Long Terminal Repeat (LTR), and 3’ LTR for vector expression. In various embodiments, the sequence of the FIV 5’LTR is SEQ ID NO: 88, and the sequence of the FIV 3’LTR is SEQ ID NO: 89.

[0225] In various embodiments, the 2STOP is a dual-function protein, comprised of a dominant-negative portion, designated DN-lirl. Wherein the DN-lirl portion of the 2STOP traps HIV particles, preventing mature virions from budding out of the membrane of HIV- infected cells. In some embodiments, the coding sequence of DN-lirl is set forth in SEQ ID NO: 45. In some embodiments, the amino acid sequence of DN-lirl is set forth in SEQ ID NO: 46.

[0226] In some embodiments, the DN-lirl SEQ ID NO: 46 comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46.

[0227] In various embodiments, the 2STOP comprises a transcriptional repressor domain. Wherein the transcriptional repressor is selected from the group consisting of Gal4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR, and functional equivalents.

[0228] In some embodiments, the nucleic acid sequence coding for the transcriptional repressor domain is selected from the group consisting of Gal4-BD SEQ ID NO: 58, GalR SEQ ID NO: 59, GalS SEQ ID NO: 60, LacI SEQ ID NO: 61, CcpA SEQ ID NO: 62, CytR SEQ ID NO: 63, Mall SEQ ID NO: 64, PurR SEQ ID NO: 65, RafR SEQ ID NO: 66, RbtR SEQ ID NO: 67, ScrR SEQ ID NO: 68, and functional equivalents thereof.

[0229] In various embodiments, the FIV cargo construct is modified to include a regulatory operator sequence placed downstream of the 5’ LTR, SEQ ID NO: 88, for auto-inhibition of the lentiviral vector’s replication. In various embodiments, the operator sequence selected for the modified wild-type FIV strand is identical to the operator sequence selected for the FIV cargo strand.

[0230] In various embodiments, the operator sequence, can include, for example, the Upstream Activation Sequence (UAS) SEQ ID NO: 58 downstream of the 5’ LTR SEQ ID NO: 88 which is specifically recognized and bound by a dimerized transcriptional repressor such as GAL4-BD SEQ ID NO: 47 and the like for transcriptional regulation or repression of downstream genes.

[0231] In various embodiments, the operator sequence is selected from a group of sequences that are specifically recognized by the cognate transcriptional repressor included in theInventor: Laura A. Prendergast2STOP protein. In various embodiments, the cognate operator sequences are selected from a list that includes SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 and functional equivalents thereof.

[0232] In various embodiments, the 2STOP protein is arranged wherein the transcriptional repressor is upstream (5’) of the dominant-negative domain. This version of the 2STOP is designated 2STOPA. The nucleic acid sequences encoding 2STOPA can, in various embodiments, be selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents thereof.

[0233] In some embodiments, the amino acid sequence of 2STOPA is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 and functional equivalents thereof.

[0234] In some embodiments, 2STOPA comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to any one of the amino acid sequences set forth from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0235] In various embodiments, the 2STOP protein is arranged wherein the dominantnegative domain is upstream (5’) of the, transcriptional repressor. This version of the 2STOP is designated 2STOPB. The nucleic acid sequences encoding 2STOPB can be selected from a list that includes: SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents thereof.

[0236] In some embodiments the amino acid sequence of 2STOPB is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and functional equivalents thereof.

[0237] In some embodiments 2STOPB comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to any one of the amino acid sequences set forth in a group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ IDInventor: Laura A. PrendergastNO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 and SEQ ID NO: 44.

[0238] In various embodiments, the nucleic acid sequence encoding 2 STOP A (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents, or 2STOPB SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents, is preceded by the HIV Transactivation Response Region (TAR sequence) SEQ ID NO: 69, which is recognized and bound by the transactivator protein (tat) delivered early in the process of HIV infection.

[0239] In various embodiments, the vector construct is comprised of the minimal sequence requirements for packaging the vector constructs into the FIV shell. The first 311 nucleotides of the native FIV gag gene, SEQ ID NO: 309, comprises the packaging signal (denoted by in the construct diagram, FIG. 1) and can be used, in some embodiments, to package the FIV genome at nearly wild-type levels to produce high-titer FIV vectors.

[0240] In various embodiments, downstream of the coding sequence for the 2STOP, the cargo construct is comprised of adjunctive protective transgenes for a multi-mechanistic approach for neutralizing the HIV infection and eradicating infectious HIV from host cells.

[0241] In various embodiments, adjunctive protective transgenes are selected from a list that includes, but is not limited to: nucleic acid aptamers, protein aptamers, short hairpin RNA (shRNA), immunologic agents, CRISPR-based gene editing technology, ribozymes, fusion inhibitors, soluble viral receptor mimics (e.g. soluble CD4), antisense oligonucleotides and the like, for knockout of HIV gene products or excision of HIV sequences from the genome.

[0242] In various embodiments, immunologic agents for targeting the HIV virus include, for example: monoclonal antibodies (mAbs), monovalent antibodies (mAbs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bispecific antibodies, engineered immuno-adhesins, functional equivalents and combinations thereof.

[0243] In conjunction with the vector described herein, auxiliary transgenes are delivered that encode immunologic agents for targeting circulating anti-HIV antibodies to accomplish seroreconversion in the recipient of the transgenes. Such auxiliary transgenes may encode, without limitation, nucleic acid aptamers or protein aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, orInventor: Laura A. Prendergast camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents, and combinations thereof.

[0244] In various embodiments, the cargo construct carries nucleic acid coding for CRISPR- associated proteins (Cas endonucleases) for CRISPR-based gene-editing. Smaller Cas orthologues have been identified that allow more efficient packaging of the Cas nuclease into a delivery vector. Small Cas orthologues include saCas9, cjCas9, sagCas9, Cas , and functional equivalents thereof. Sequences encoding smaller Cas orthologs are selected from a list that includes Staphylococcus aureus-derived saCas9 (3.16 kb) SEQ ID NO: 92, Campylobacter jejuni -derived cjCas9 (2.95 kb) SEQ ID NO: 93, Staphylococcus agnetis- derived sagCas9 (2.98 kb) SEQ ID NO: 94, Cas , SEQ ID NO: 95 and functional equivalents.

[0245] In various embodiments, downstream of the 2STOP coding sequence, the cargo construct carries sgRNA sequences for CRISPR-based site-specific integration of the vector’s genome into safe harbor loci in the host genome. sgRNA sequences can be designed by means of design tools known to persons of skill in the art. See, for example, Cui, Y., Xu, J., Cheng, M., Liao, X., & Peng, S. (2018). Review of CRISPR / Cas9 sgRNA Design Tools. Interdisciplinary Sciences: Computational Life Sciences, 10(2), 455-465. See also: Chuai, G., Wang, Q., & Liu, Q. (2016). In Silico Meets in vivo: Towards Computational CRISP R- Based sgRNA Design. Trends in Biotechnology, 1-10.

[0246] In various embodiments the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV gag-pol by insertion of the vector construct into the coding sequence for HIV gag-pol. In some embodiments, the sgRNA sequence for knockout of gag- pol are selected from a list that includes SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO:116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO:121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO:126, SEQ ID NO: 127, SEQ ID NO: 128, and the like.

[0247] In various embodiments the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV integrase by insertion of the vector construct into the coding sequence for HIV integrase. In some embodiments, the sgRNA sequence for insertion into HIV integrase is selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 130,Inventor: Laura A. PrendergastSEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and the like.

[0248] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV env by insertion of the vector construct into the coding sequence for HIV env. In some embodiments, the sgRNA sequence for insertion into HIV env are selected from the group consisting of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and the like.

[0249] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV vif by insertion of the vector construct into the coding sequence for HIV vif. In some embodiments, the sgRNA sequence for insertion into HIV vif are selected from the group consisting of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and the like.

[0250] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV vpu by insertion of the vector construct into the coding sequence for HIV vpu. In some embodiments, the sgRNA sequence for insertion into HIV vpu are selected from a list that includes SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, and the like.

[0251] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV vpr by insertion of the vector construct into the coding sequence for HIV vpr. In some embodiments, the sgRNA sequence for insertion into HIV vpr are selected from the group consisting of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, and the like.

[0252] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV rev by insertion of the vector construct into the coding sequence for HIV rev. In some embodiments, the sgRNA sequence for insertion into HIV rev are selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192 and the like.Inventor: Laura A. Prendergast

[0253] In various embodiments, the cargo construct carries sgRNA sequences for CRISPR- mediated knockout of HIV vpx by insertion of the vector construct into the coding sequence for HIV vpx. In some embodiments, the sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpx sequence are selected from the group consisting of SEQ ID NO. 193, SEQ ID NO. 194, and the like.

[0254] In some embodiments, the CRISPR-based transfer of the lentiviral vector’s construct into the host genome makes use of sgRNA targeting the human CCR5 receptor for insertional disruption of the receptor most commonly used by HIV for entry into host cells. In various embodiments the sgRNA sequences for insertion into the human CCR5 receptor are selected from the group consisting of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209 and the like. See for example, U.S. Patent No. 10,745,677 which describes the use of CRISPR-based gene editing technology for introducing genetic modifications that result in loss of function in the CCR5 gene. U.S. Patent No. 10,745,677 is herein incorporated by reference. See also U.S. Patent Publication No. US 2020 / 0392487, which describes excision of retroviral nucleic acid sequences by means of CRISPR-based gene editing. US Patent Publication No. US 2020 / 0392487 is herein incorporated by reference.

[0255] In various embodiments, the CRISPR-based gene editing technology directs excision of HIV sequences from the genome of infected host cells (See e.g., Kaminski, R., Chen, Y., Fischer, T., Tedaldi, E., Napoli, A., Zhang, Y., Kam, J., Hu, W ., & Khalili, K. (2016). Elimination ofHIV-1 Genomes from Human T-lymphoid Cells by CRISPR / Cas9 Gene Editing. Nature Publishing Group, December 2015, 1-15. See also: Bhowmik, R., & Chaubey, B. (2022). CR1SPR / Cas9: a tool to eradicate HIV-L AIDS Research and Therapy, 1-14).

[0256] In some embodiments, constructs as described herein comprise more than one promoter (e.g., 2, 3, 4, 5, or more promoters). Examples of promoters that can be used in various embodiments described herein include, for example, promoters that are native to FIV such as the 5’ LTR, SEQ ID NO: 88, and the 3’ LTR, SEQ ID NO: 89, for replication of vector sequences.Inventor: Laura A. Prendergast

[0257] In some embodiments, one or more of the promoters in a construct described herein is a constitutive promoter. In various embodiments, constitutive promoters are used to drive expression of the 2 STOP gene product.

[0258] In some embodiments, one or more of the promoters in a construct described herein is a cell-type specific promoter which drives expression of operably linked coding sequences in a specific cell or tissue type, as a function of each cell’s unique collection of transcriptional machinery components that interact with the cell-type specific promoter. Wherein the celltype specific promoter is selected from a list that includes: CD4, CD8, and CD34, and the like.

[0259] Provided herein, in various embodiments are therapeutic genes, or transgenes, that encode proteins with an anti-HIV function. Transgenes that can be introduced, in various embodiments described herein, include 2STOPA, 2STOPB, mAbs, bNmAbs, Fabs, anti- idiotypic antibodies, camelid nanobodies, nucleic acid aptamers, protein aptamers, shRNA that can be processed into RNAi for knockdown of HIV gene products, CRISPR-associated endonucleases (Cas) with signal guide RNA (gRNA) sequences for site-specific insertion of lentivector constructs into HIV coding sequences, or for excision of HIV from the infected host’s genome, and the like.

[0260] 2STOPA, in various embodiments, is encoded by a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 and functional equivalents thereof.

[0261] 2STOPB, in various embodiments, is encoded by a sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33 and functional equivalents thereof.

[0262] In some embodiments, the adjunctive protective transgenes comprise short-hairpin RNA (shRNA) sequences targeting expressed HIV gene products. As used herein, a person of ordinary skill in the art would prepare short hairpin RNA (shRNA) sequences for the knockout of expressed proteins by first identifying a 19-22 nucleotide target sequence within the mRNA transcript of the gene of interest, typically located downstream of the start codon and in regions of moderate GC content (40-60%) to optimize stability and efficacy. Target sequences are selected to avoid significant homology with other genes in the host genome, thereby minimizing off-target effects. Once candidate sequences are identified,Inventor: Laura A. Prendergast complementary sense and antisense strands are designed, separated by a short loop sequence (commonly 4-11 nucleotides, e.g., TTCAAGAGA), and configured to form a hairpin structure upon transcription. Upon introduction into host cells, the shRNA is processed by the endogenous RNA interference (RNAi) pathway into small interfering RNA (siRNA), leading to sequence-specific degradation of the target mRNA and functional gene knockout.

[0263] In certain embodiments, the design of short hairpin RNA (shRNA) sequences may be facilitated by persons of skill in the art through the use of publicly available or commercially accessible online sequence design tools that are configured to identify optimal target sequences within a given messenger RNA (mRNA) transcript while minimizing off-target effects. Non-limiting examples of such tools include the BLOCK-iT™ RNAi Designer and can be obtained from a variety of sources, for example, ThermoFisher Scientific, which provides automated siRNA and shRNA sequence prediction with genome-wide specificity filtering; the siDESIGN Center (Horizon Discovery;), which employs proprietary algorithms for identifying optimal RNA interference (RNAi) target sites; the siRNA Wizard which facilitates loop sequence optimization and GC content adjustment for shRNA constructs; and the SplashRNA platform (Broad Institute), which utilizes validated prediction models for the selection of potent shRNA sequences in mammalian systems. These tools may be employed individually or in combination to guide the rational design of shRNA constructs suitable for experimental or therapeutic applications.

[0264] In various embodiments, the expressed HIV gene products to be targeted are selected from gag-pol, integrase, env, vif, vpu, vpr, rev, or combinations thereof. In further embodiments, sequences derived from HIV-2 or related lentiviruses, such as vpx, may also be targeted for disruption.

[0265] In some embodiments, the shRNA sequences for gag-pol knockout are selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO:218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO:223, SEQ ID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO:228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO:233, and functional equivalents thereof.

[0266] In some embodiments, the shRNA sequences for integrase knockout are selected from the group consisting of SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, and functional equivalents thereof.Inventor: Laura A. Prendergast

[0267] In some embodiments, the shRNA sequences for env knockout are selected from the group consisting of SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, and functional equivalents thereof.

[0268] In some embodiments, the shRNA sequences for vif knockout are selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, and functional equivalents thereof.

[0269] In some embodiments, the shRNA sequences for vpu knockout are selected from the group consisting of SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, and functional equivalents thereof.

[0270] In some embodiments, the shRNA sequences for vpr knockout are selected from the group consisting of SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, and functional equivalents thereof.

[0271] In some embodiments, the shRNA sequences for rev knockout are selected from the group consisting of SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQ ID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274,SEQ ID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279,SEQ ID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284,SEQ ID NO: 285, and functional equivalents thereof.

[0272] In some embodiments, the shRNA sequences for vpx knockout are selected from the group consisting of SEQ ID NO. 286, SEQ ID NO. 287, SEQ ID NO. 288, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 291, SEQ ID NO. 292, SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 295, and functional equivalents thereof.

[0273] In various embodiments described herein, the lentiviral vector also comprises auxiliary transgenes for targeting circulating HIV antibodies to accomplish seroreconversion, wherein the auxiliary transgenes encode nucleic acid aptamers or protein aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, or camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents; and combinations thereof,.Inventor: Laura A. Prendergast

[0274] In various embodiments, the FIV vector is pseudotyped by inclusion of a sequence coding for a heterologous coat protein such as VSV-G, SEQ ID NO: 296, to provide broader host tropism.

[0275] In various embodiments, the heterologous envelope protein for pseudo-typing the vector can be from a variety of enveloped viruses, including, for example: Vesicular Stomatitis Virus (VSV), Lymphocytic choriomeningitis virus (LCMV), Alphavirus Ross River virus (RRV), Marburg virus, Lassa virus, Baculovirus GP64 (GP64), and functional equivalents thereof.

[0276] In some embodiments, the nucleic acid sequences encoding the heterologous coat protein for pseudotyping can, in various embodiments, be selected from the group consisting of VSV-G, SEQ ID NO: 296 LCMV, SEQ ID NO: 297, RRV, SEQ ID NO: 298, MARVGP, SEQ ID NO: 299 LASVGP, SEQ ID NO: 300, GP64, SEQ ID NO: 301, functional equivalents, and combinations thereof.

[0277] In some embodiments, the amino acid sequences of the heterologous envelope protein can be selected from a group consisting of, for example, VSV-G, SEQ ID NO: 302, Lymphocytic choriomeningitis virus (LCMV), SEQ ID NO: 303, Alphavirus Ross River virus (RRV), SEQ ID NO: 304, Marburg virus, SEQ ID NO: 305, Lassa virus, SEQ ID NO: 306, Baculovirus GP64 (GP64), SEQ ID NO: 307, functional equivalents and combinations thereof.

[0278] In various embodiments, FIV vector particles can be prepared, purified and concentrated, such that the FIV vector particle is capable of infecting mammalian cells in vivo. Representative methods for concentrating and / or purifying vector particles include centrifugation, precipitation (e.g., utilizing PEG), filtration, and column chromatography using a protocol such as that described in Benskey, M. J., & Manfredsson, F. P. (2017). Chapter 8 Lentivirus Production and Purification.

[0279] In various embodiments, FIV vector particles that have been are purified or concentrated are preserved by first adding a sufficient amount of a formulation buffer to the media containing the FIV vector particles, in order to form an aqueous suspension. The formulation buffer is an aqueous solution that contains a saccharide, a high molecular weight structural additive, and a buffering component in water. The aqueous solution may also contain one or more amino acids.

[0280] As noted above, high titer recombinant lentiviral particles of the present invention can be administered to a wide variety of locations including, for example, into sites such asInventor: Laura A. Prendergast the cerebral spinal fluid, bone marrowjoints, arterial endothelial cells, rectum, buccal / sublingual, vagina, the lymph system, to an organ selected from the group consisting of lung, liver, spleen, skin, blood and brain, or to a site selected from the group consisting of tumors and interstitial spaces. Within other embodiments, the FIV vector particle may be administered intraocularly, intranasally, sublingually, orally, topically, intravesically, intrathecally, topically, intravenously, intraperitoneally, intracranially, intramuscularly, or subcutaneously. Other representative routes of administration include gastroscopy, ECRP and colonoscopy, which do not require full operating procedures and hospitalization, but may require the presence of medical personnel.

[0281] In various embodiments the method of introducing into recipient is intravenous, or intranasal. Viral particles are concentrated, and delivered in a PBS or DPBS solution.(Condiotti, R., Curran, M. A., Nolan, G. P., Giladi, H., Ketzinel-Gilad, M., Gross, E., & Galun, E. (2004). Prolonged liver-specific transgene expression by a non-primate lentiviral vector. Biochemical and Biophysical Research Communications, 320(3), 998-1006).(Carbonaro-Sarracino, D. A., Tarantal, A. F., Lee, C. C. I., Kaufman, M. L., Wandro, S., Jin, X., Martinez, M., Clark, D. N., Chun, K., Koziol, C., Hardee, C. L., Wang, X., & Kohn, D. B. (2020). Dosing and Re-Administration of Lentiviral vector for In vivo Gene Therapy in Rhesus Monkeys and ADA-Deficient Mice. Molecular Therapy - Methods and Clinical Development, 76(March), 78-93).

[0282] Many of the routes of administration described herein (e.g., into the CSF, into bone marrow, into joints, intravenous, intra-arterial, intracranial intramuscular, subcutaneous, into various organs, intra-tumor, into the interstitial spaces, intra-peritoneal, intra-lymphatic, or into a capillary bed) may be accomplished simply by direct administration using a needle, catheter or related device. In particular, within certain embodiments of the invention, one or more dosages may be administered directly in the indicated manner at dosages greater than or equal to 103, 104, 105, 106, 107, 108, 109, 1010or 1011colony forming units (cfu).

[0283] Efficacy of treatment can be assayed by measuring the usual indicators of disease progression, including antibody level, viral antigen production, infectious HIV levels, or levels of nonspecific infections.REGULATORY ELEMENTS OF THE LENTIVIRAL VECTOR

[0284] In some embodiments, the lentiviral-based vector comprises a 5' LTR SEQ ID NO: 88 containing cis-acting promoter and enhancer sequences configured to drive transcription of the packaged genome. The 5' LTR SEQ ID NO: 88 of feline immunodeficiency virusInventor: Laura A. Prendergast(FIV) functions as the principal promoter region that regulates the initiation of viral transcription. Upon infection, the 5' LTR SEQ ID NO: 88 is recognized by the host’s RNA polymerase II machinery, leading to the transcription of the integrated provirus. Additionally, the 5' LTR SEQ ID NO: 88 plays a role in the early steps of reverse transcription and integration, acting as a key regulatory element that coordinates temporal expression of viral genes. In lentiviral vector systems, the 5' LTR SEQ ID NO: 88 may be modified to include exogenous promoter elements or to incorporate self-inactivating (SIN) mutations that reduce basal transcriptional activity after integration.

[0285] In certain embodiments, the 3' LTR SEQ ID NO: 89 of FIV serves as a critical sequence in the reverse transcription process, where it is duplicated to form the new 5' LTR SEQ ID NO: 88 during proviral DNA synthesis. While the 3' LTR SEQ ID NO: 89 does not directly drive transcription in the integrated provirus, it plays a pivotal role in ensuring proper termination and polyadenylation of viral transcripts. It contains regulatory elements necessary for transcriptional termination and efficient processing of viral mRNA. Furthermore, in the context of vector design, the 3' LTR SEQ ID NO: 89 can be engineered to include deletions or mutations that confer self-inactivation, thereby reducing the risk of insertional mutagenesis or aberrant transcriptional activation from the integrated vector. This makes the 3' LTR SEQ ID NO: 89 a valuable site for vector safety modifications in FIV-based gene delivery platforms.

[0286] In some embodiments, expression of the 2STOP transgene is driven by a constitutive promoter. Constitutive promoters may be selected from a list that includes hCMV, EFlalpha, CAGG, SV40, and functional equivalents thereof. In some embodiments, the constitutive promoter sequence is selected from the group consisting of hCMV SEQ ID NO: 81, EF-1 alpha SEQ ID NO: 82, CAGG SEQ ID NO: 83 and SV40 SEQ ID NO: 84, and functional equivalents thereof, all of which are commonly used in mammalian systems. Driving expression of the 2STOP from a constitutive promoter is desirable for establishing inhibition of FIV replication in all cells receiving the transgenes until challenge with infectious HIV.

[0287] In certain embodiments, the FIV cargo construct is engineered to incorporate a regulatory operator sequence positioned downstream of the 5 ' LTR, SEQ ID NO: 88, to enable auto-inhibitory control over replication of the lentiviral vector. In some embodiments, the operator sequence incorporated into the modified wild-type FIV strand is identical to the operator sequence incorporated into the FIV cargo strand.Inventor: Laura A. Prendergast

[0288] In some embodiments, operator sequences can be selected from the group consisting of sequences recognized by the following transcriptional repressors: Gal4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR, and the like. In some embodiments, the operator sequence is selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and functional equivalents thereof.

[0289] In various embodiments, presence of infectious HIV transiently relieves inhibition of vector replication by recruiting 2STOP SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44 and functional equivalents, away from the operator sequence placed downstream of the 5’ LTR SEQ ID NO: 88 in both the modified wild-type and cargo vector constructs, through interaction of the p6-lirl domain of the 2STOP with budding virions.

[0290] In certain embodiments, the FIV-based lentivector can be placed under the control of a cell- or tissue-specific promoter, thereby limiting expression of the FIV lentivector sequences to those specific cells or tissues within which the promoter is active. In various embodiments, the cell-type specific promoter is selected from the group consisting of CD4, CD8, and CD34, and functional equivalents thereof. In various embodiments, the sequence of the cell-type specific promoter is selected from the list that includes: CD4, SEQ ID NO: 85, CD8, SEQ ID NO: 86, CD34, SEQ ID NO: 87, and functional equivalents thereof.MULTI-MECHANISTIC ANTI-HIV GENETIC STRATEGIES

[0291] In addition to the regulatory elements described above, in various embodiments, the lentiviral vector constructs for use herein also comprise one or more adjunctive protective transgenes for a multi -mechanistic approach to eradicating infectious HIV from host cells.

[0292] In various embodiments, the FIV-based lentivector comprises nucleic acid sequences encoding CRISPR-associated nucleases (Cas proteins) in conjunction with signal guide RNA (sgRNA) that can be used for site-specific insertion of the lentivector’ s genetic constructs. sgRNA for gene-editing applications may be designed by a person of skill in the art using available sgRNA design tools. See, for example, Wang, J., Zhang, X., Cheng, L., & Luo, Y.Inventor: Laura A. Prendergast(2020). An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools. RNA Biology, 17(1), 13-22.

[0293] In some embodiments, the CRISPR-based gene editing technology can be used for introducing the vector’ s transgenes into HIV coding sequences (knock-in) within the genome of the infected subject, thereby disrupting the HIV-encoded sequences.

[0294] In some embodiments, the HIV coding sequences that might be disrupted by use of CRISPR-based site-specific integration are selected from the group consisting of HIV gag- pol, integrase, env, vi vpu, vpr, rev, vpx, and the like.

[0295] In various embodiments, CRISPR-based gene editing can be used to insert vector sequences into the human T-cell CCR5 receptor to accomplish a genetic knockout.

[0296] In various embodiments, CRISPR-based gene editing can be used for excision of HIV sequences from the subject’s genome.

[0297] In various embodiments, the FIV-based lentivector may be used for delivery of transgenes coding for short-hairpin RNA (shRNA), nucleic acid aptamers, protein aptamers, and the like, for targeted knockout of HIV encoded proteins.

[0298] In various embodiments, the adjunctive protective transgenes also comprise immunologic-based approaches for targeting infectious HIV virions, wherein the immunologic-based approaches comprise, for example, monoclonal antibodies (mAbs), monovalent antibodies (mAbs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bispecific antibodies, engineered immuno-adhesins, functional equivalents and combinations thereof.

[0299] In addition to the anti-HIV approaches described above, in various embodiments, the lentiviral vector cargo strand may encode auxiliary transgenes to accomplish seroreconversion, wherein the auxiliary transgenes are selected from a group that encodes, for example, expression of nucleic acid aptamers; protein aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, or camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents; and combinations thereof.IMMUNOLOGIC-BASED APPROACHES FOR NEUTRALIZING HIV

[0300] In some embodiments, the methods described herein include the use of monoclonal antibodies directed against CD4 (mAb-CD4) to inhibit HIV entry into CD4+T lymphocytes.Inventor: Laura A. PrendergastSuch antibodies are capable of interfering with HIV binding by blocking viral access to uninfected CD4+cells. In certain embodiments, the method further comprises administering a soluble, secreted form of CD4 that lacks the cytoplasmic and transmembrane domains, wherein the truncated CD4 retains high-affinity binding to the HIV envelope glycoprotein gpl20, with an affinity comparable to that of full-length CD4. The binding of this soluble CD4 to gpl20 neutralizes HIV infectivity. (See, e.g., Moroz, V. I., Zh, P., Smith, D. H., Bymi, R. A., Marsters, S. A., Gregory, T., Groopman, J. E., & Capon, D. J. (1984). Blocking of HIV-1 Infectivity by a Soluble, Secreted Form of the CD4 Antigen. Science 238 (vol . 1966), pp 1982-1985).

[0301] While administration of soluble CD4 protein has demonstrated antiviral activity, its clinical application has proven impractical due to challenges in achieving therapeutically effective concentrations in vivo. Accordingly, in some embodiments, the present disclosure provides methods in which a nucleic acid sequence encoding a soluble form of CD4 is introduced into target cells and expressed under the control of a strong internal promoter. This approach facilitates sustained, cell-based production of soluble CD4 at levels sufficient to achieve functional neutralization of HIV infectivity. (See, e.g., R. A. Morgan, D. J. Looney, D. D. Muenchau, F. Wong-Staal, R. C. Gallo & W. F. Anderson, Retroviral vectors expressing soluble CD 4: a potential gene therapy approach for HIV infection, AIDS Res Hum Retroviruses, 1990;6(l 1): 183-189).

[0302] In some embodiments, the methods described herein include the use of monoclonal antibody CG10, which is capable of inhibiting HIV-1 infection by blocking the interaction between the viral envelope glycoprotein gpl20 and its required cellular co-receptor. In certain embodiments, enhanced neutralization is achieved by fusing the single-chain variable fragment (scFv) of CG10 with the CD4 molecule, thereby generating a bifunctional chimeric polypeptide with increased anti-HIV-1 activity. In further embodiments, this chimeric construct is fused to the Fc region of human immunoglobulin G1 (IgGl), thereby improving stability, half-life, and effector function. (See, e.g., Meyuhas, R., Noy, H., Montefiori, D. C., Denisova, G., Gershoni, J. M., & Gross, G. (2005). HIV-1 neutralization by chimeric CD4- CG10 polypeptides fused to human IgGl. 42, 1099-1109, incorporated herein by reference).

[0303] In various embodiments, the immunologic agents for targeting HIV comprise anti -idiotypic antibodies capable of eliciting a protective, endogenous anti-HIV immune response in the subject. Anti -idiotypic antibodies function as structural mimics of HIV-specific antigens, including but not limited to epitopes located on the HIV envelopeInventor: Laura A. Prendergast glycoproteins gpl20 and gp41, thereby inducing the host immune system to generate antibodies that recognize and neutralize authentic HIV virions. In certain embodiments, the anti -idiotypic antibodies are engineered to enhance immunogenicity, increase antigenic mimicry fidelity, or improve stability in vivo. Administration of such anti -idiotypic antibodies may result in a sustained anti-HIV humoral response, providing both therapeutic and prophylactic benefits.

[0304] In some embodiments, anti-Id antibody HR1.7 is raised against a monoclonal antibody (mAb) that targets CD4, which can partially neutralize HIV infection of human T- cells in vitro (See e.g., Chanh, T. C., Dreesman, G. R., & Kennedy, R. C. (1987). Monoclonal anti-idiotypic antibody mimics the CD4 receptor and binds human immunodeficiency virus. Proc. Natl. Acad. Sci. 84(June), 3891-3895).

[0305] In some embodiments, the methods described herein employ the approach disclosed in U.S. Pat. Nos. 6,057,421 and 6,768,004, each of which is incorporated by reference in its entirety. These patents describe the use of monoclonal antibody 1F7, an anti -idiotypic antibody reactive with anti-HIV antibodies, for treating HIV infection. In certain embodiments, the method comprises administering the 1F7 antibody or a nucleotide sequence encoding the 1F7 antibody in vivo to stimulate broadly reactive anti-HIV antibody production and activate HIV-specific, committed B cells, thereby functioning as an effective therapeutic vaccine.

[0306] In some embodiments, the present disclosure provides additional immunologic-based therapies comprising fusion antibodies that concurrently target the CD4 receptor and the HIV-1 envelope glycoprotein. Such fusion antibodies exhibit enhanced potency and breadth of neutralization relative to monospecific anti-HIV antibodies. In certain embodiments, these fusion constructs are designed to engage both viral and host targets, thereby improving therapeutic efficacy. Fusion antibodies of this nature are described in U.S. Pat. No.8,637,024, which is hereby incorporated by reference in its entirety.

[0307] In some embodiments, the methods and compositions described herein involve the use of broadly neutralizing monoclonal antibodies (bNmAbs) directed against HIV-1. A rare subset of HIV-l-infected individuals, referred to as "elite neutralizers," naturally develop bNmAbs capable of neutralizing over 80% of circulating HIV-1 strains. Advances in antibody discovery technologies have facilitated the identification and characterization of multiple bNmAbs from such individuals, each exhibiting distinct specificities toward conserved epitopes on the HIV-1 envelope glycoprotein (Env). See Jaworski, J. P., & Cahn,Inventor: Laura A. PrendergastP. (2018). Review. Preventive and therapeutic features of broadly neutralizing monoclonal antibodies against HIV- 1. The Lancet HIV, 5(12), e723-e731).

[0308] In some embodiments, the present disclosure provides methods for isolating broadly neutralizing antibodies (bNAbs) against HIV-1, as described in U.S. Pat. No. 10,407,493, which is hereby incorporated by reference in its entirety. The method comprises screening memory B cell cultures derived from donor peripheral blood mononuclear cell (PBMC) samples for neutralization activity against a panel of diverse HIV-1 strains. A memory B cell exhibiting broad-spectrum neutralizing activity is selected and cloned. A monoclonal antibody is subsequently rescued and expressed from the selected memory B cell culture.

[0309] In some embodiments, the present disclosure provides methods for obtaining broadly neutralizing antibodies (bNAbs) capable of targeting a plurality of HIV-1 strains, as described in U.S. Pat. No. 10,934,345, which is hereby incorporated by reference. The referenced patent discloses techniques for identifying and isolating human monoclonal antibodies with broad-spectrum neutralizing activity through screening of antigen-specific B cells, followed by molecular cloning and characterization of antibody sequences. Such methods facilitate the development of potent therapeutic candidates and inform rational vaccine design by targeting conserved epitopes across divergent HIV-1 variants.

[0310] Numerous patent documents describe the development and characterization of monoclonal antibodies exhibiting broad specificity for conserved HIV epitopes such as, for example, U.S. Pat. Nos. 10,519,222; 10,836,811; 10,093,720; and 10,865,234, and U.S. Patent Publication Nos. 2019 / 0016786, 2016 / 0289305, and 2022 / 0202956, which are herein incorporated by reference in their entirety.

[0311] In some embodiments, numerous additional immunologic-based anti-HIV strategies that can be delivered include, for example: hematopoietic stem cell transplantation, to equip infected subjects with T-cells lacking the CCR5 receptor; the “shock and kill” approach, which uses pharmacological agents to trigger proliferation of HIV in infected cells, coupled with therapeutic vaccines or monoclonal antibodies to destroy infected cells; blockade of immune checkpoint molecules to restore T-cell function; and immune-modulating drugs to promote T cell activation and proliferation, reviewed in Barouch, D. H., & Deeks, S. G. (2014). Immunologic strategies for HIV- 1 remission and eradication. Science, 345(6193), 169-174.

[0312] In some embodiments, ex vivo autologous chimeric antigen receptor T-cell (CAR-T) therapies are employed to target HIV-infected cells. T-cells are isolated from the subject,Inventor: Laura A. Prendergast engineered ex vivo to express CARs specific for HIV antigens such as gpl20 or gp41, expanded, and re-infused into the subject. The CAR-T cells recognize and destroy infected cells in an MHC-independent manner and may incorporate co-stimulatory domains (e.g., CD28, 4-1BB) to enhance function. In certain embodiments, CAR-T cells are further modified, for example by CCR5 disruption, to confer resistance to HIV reinfection and provide durable antiviral activity after a single administration.

[0313] In certain embodiments, the lentiviral gene delivery system is administered in conjunction with immunologic interventions to enhance antiviral immunity. Such immunologic interventions include monoclonal antibodies, including broadly neutralizing antibodies (bNAbs) specific for HIV envelope glycoproteins; fusion antibodies configured to simultaneously target multiple HIV or host-cell epitopes; therapeutic vaccines designed to elicit HIV-specific humoral and / or cellular immune responses; and immune-modulatory agents such as checkpoint inhibitors, toll-like receptor agonists, and cytokines. In such combinations, the lentiviral vector confers a durable, cell-intrinsic antiviral function that complements immune-based mechanisms of action, thereby increasing the probability of functional or sterilizing cure.APTAMER-BASED THERAPIES

[0314] In some embodiments, the present disclosure provides aptamers — synthetic high- affinity ligands composed of single-stranded nucleic acids — that are capable of binding target molecules involved in specific HIV-related functions via non-covalent interactions. Aptamers exhibit molecular recognition capabilities comparable to those of antibodies, yet offer distinct advantages in terms of ease of engineering, low immunogenicity, and versatility in therapeutic design. Their broad range of potential applications renders them highly suitable for use in antiviral gene therapies. Aptamer design may be guided using established in vitro selection strategies, including those described by Vorobyeva MA, Davydova AS, Vorobjev PE, Venyaminova AG. Key Aspects of Nucleic Acid Library Design for in vitro Selection. Int J Mol Sci. 2018 Feb 5;19(2):470, which outlines critical considerations for nucleic acid library construction and optimization.

[0315] In some embodiments, the present disclosure provides protein aptamers — synthetic polypeptide ligands engineered to bind HIV-related targets with high specificity and affinity via non-covalent interactions. Comparable to monoclonal antibodies in molecular recognition yet offering advantages such as reduced size, ease of intracellular expression, and access to otherwise inaccessible protein-protein interfaces, protein aptamers may be designed orInventor: Laura A. Prendergast selected using combinatorial display technologies (e.g., yeast, phage, or ribosome display) to identify optimized binding domains (see, e.g., Colas P, Cohen B, Jessen T, Grishina I, McCoy J, Brent R. Genetic selection of peptide aptamers that recognize and inhibit cyclin- dependent kinase 2. Nature. 1996 Apr 11;380(6574):548-50). Their structural versatility and favorable biochemical properties make them well-suited for incorporation into antiviral gene therapy vectors.

[0316] In some embodiments, aptamers are directed against the HIV envelope glycoprotein gpl20, thereby inhibiting its interaction with host cell surface receptors and preventing viral entry. The binding of gpl20 to the CD4 receptor is a critical step in HIV-1 cell entry and membrane fusion, making this interaction a strategic target for anti-HIV therapeutics. Aptamers that disrupt this gpl20-CD4 interface may serve as effective agents for blocking initial stages of viral infection.

[0317] In some embodiments, anti-HIV aptamers are employed to inhibit key stages of the viral life cycle, including viral entry, reverse transcription, proteolytic processing, chromosomal integration, viral gene expression, and virion packaging. Aptamers may be designed to bind viral enzymes or structural components, thereby interfering with essential HIV functions. Exemplary molecular targets include the Rev / RRE and Tat / TAR regulatory elements, as well as viral proteins such as p55Gag, matrix, and nucleocapsid. Such aptamers have been shown to reduce viral replication and virion production, as described in Held DM, Kissel JD, Patterson JT, Nickens DG, Burke DH. HIV-1 inactivation by nucleic acid aptamers. Front Biosci. 2006 Jan 1; 11 : 89- 112; and Ramalingam, D., Duclair, S., Datta, S. A. K., Ellington, A., Rein, A., & Prasad, V. R. (2011). RNA Aptamers Directed to Human Immunodeficiency Virus Type 1 Gag Polyprotein Bind to the Matrix and Nucleocapsid Domains and Inhibit Virus Production. S5(l), 305-314).

[0318] In some embodiments, anti-HIV strategies include the use of chimeric constructs comprising a gpl20-targ eting aptamer linked to a small interfering RNA (siRNA). Such aptamer-siRNA chimeras enable cell-type-specific delivery of gene-silencing agents, thereby enhancing therapeutic precision and efficacy. Notably, this approach has demonstrated promise as a targeted antiviral strategy, as described by Zhou, J., Li, H., Zhang, J., Piotr, S., & Rossi, J. (2011). Development of Cell-type specific anti-HIV gpl20 aptamers for siRNA delivery. June, 1-11. shRNA FOR GENE KNOCKOUTInventor: Laura A. Prendergast

[0319] In some embodiments, short hairpin RNA (shRNA) sequences are employed to silence HIV gene expression through RNA interference (RNAi). When expressed intracellularly, shRNA is processed in the cytoplasm by the Dicer enzyme into short interfering RNA (siRNA) duplexes. These duplexes are subsequently incorporated into the RNA-Induced Silencing Complex (RISC), which guides sequence-specific degradation of complementary HIV mRNA targets. This mechanism enables efficient post-transcriptional gene silencing of critical viral coding sequences. shRNA molecules may be rationally designed using bioinformatic tools and design principles, such as those reviewed by Ros, X. B., & Gu, S. (2023). Guidelines for the optimal design of miRNA-based shRNAs. METHODS, April 2016, to optimize potency, specificity, and stability.

[0320] In some embodiments, shRNA molecules are designed to target conserved coding regions within the HIV genome, thereby suppressing viral replication through RNA interference. In certain embodiments, shRNA-mediated silencing is directed against one or more HIV-1 coding sequences. Target sequences may include gag-pol (encoding the structural proteins MA, CA, NC, and p6, as well as the enzymatic proteins PR, RT, RNase H, and IN), env (encoding the envelope glycoproteins gpl20 and gp41), and regulatory elements such as tat and rev. Additional targets include accessory genes such as nef vif vpr, and vpu. In some embodiments, sequences derived from HIV-2 or other related lentiviruses, including vpx, may likewise be incorporated or subject to silencing.CONSTRUCTION OF LENTIVIRAL GENE DELIVERY VECTORS

[0321] The vector in various embodiments herein, can be prepared by persons of skill in the art. Within the main embodiment, a lentiviral gene delivery vehicle (vector) is provided which is constructed to carry or express selected genes and sequences of interest. Briefly, the FIV lentiviral vector may be readily constructed by a person with skill in the art, using techniques of genetic engineering such as might be found in a laboratory handbook on genetic engineering (e.g. Sambrook, J., Fritsch, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; or Vennison, S. J., Laboratory Manual for Genetic Engineering, 1st ed., 2009). Such retroviruses may be readily obtained from the NIH’s HIV Reagent Program Cell lines required for development or production of the lentiviral vector can be obtained from depositories or collections such as the American Type Culture Collection.

[0322] Reference is now made to FIG. 1, which depicts an exemplary schematic map of the feline immunodeficiency virus (FlV)-based lentiviral vector cargo construct ofInventor: Laura A. Prendergast the present disclosure. In various embodiments, the FIV cargo construct is engineered in accordance with the configuration shown in FIG. 1 to comprise the principal cis-acting elements required for efficient packaging, reverse transcription, and integration, together with one or more heterologous expression cassettes configured to deliver therapeutic or prophylactic nucleic acids to target cells. The relative positions and orientations of the regulatory sequences, structural features, and functional domains are indicated for illustrative purposes and are not drawn to scale. It will be appreciated by those skilled in the art that modifications to the sequence composition, length, or arrangement of these elements may be implemented without departing from the intended functional characteristics or the scope of the present disclosure.

[0323] In various embodiments, the lentiviral vector is packaged in a feline immunodeficiency virus (FIV) shell. FIV vectors can be readily constructed from a wide variety of FIV strains. Representative strains that can be used include, for example, the Petaluma isolate, the San Diego isolate, the FIV isolate 30, the FIV isolate 26, the FIV subtype C and the like. In various embodiments, the sequences of the FIV genome for engineering the vector may be selected from the Petaluma isolate, SEQ ID NO: 329, the San Diego isolate, SEQ ID NO: 330, the FIV isolate 30, SEQ ID NO: 331, the FIV isolate 26, SEQ ID NO: 332, the FIV subtype C, SEQ ID NO: 333 and functional equivalents thereof. FIV strains can either be obtained from feline isolates, from depositories or collections such as the ATCC, or isolated from known sources using commonly techniques known to persons of skill in the art.

[0324] In some embodiments, any of the aforementioned feline immunodeficiency virus (FIV) strains may be employed in the assembly of lentiviral -based gene delivery vehicles, utilizing standard recombinant DNA techniques and the teachings of the present disclosure. The modular nature of the lentiviral vector system permits the integration of functional elements derived from multiple viral sources. Accordingly, in certain embodiments, components of the FIV-based gene delivery vehicle — such as structural, regulatory, or packaging sequences — may be substituted or supplemented with analogous elements from heterologous lentiviruses or other viral backbones, provided functional compatibility is maintained. This flexible design framework enables the construction of optimized vectors tailored for specific therapeutic or experimental applications.

[0325] In some embodiments, lentiviral vector constructs are engineered to permit the expression of multiple genes of interest from a single transcript. This may be achievedInventor: Laura A. Prendergast through the use of oligoci str onic expression cassettes, in which individual coding sequences are arranged in tandem and separated by short intervening sequences — typically 120 nucleotides or fewer. Such configurations enable coordinated expression of multiple transgenes within a single host cell, facilitating the delivery of complex therapeutic payloads or combinatorial genetic interventions.

[0326] In various embodiments, the lentiviral vector is comprised of two plus-stranded RNA genomic constructs. The first construct is designated the “modified wild-type construct,” and the second construct is referred to as the “cargo construct.”

[0327] In various embodiments, both the modified wild-type and therapeutic cargo constructs comprise a 5' Long Terminal Repeat (LTR), SEQ ID NO: 88, and a 3' FIV-derived LTR, SEQ ID NO: 89. These LTR sequences contain essential regulatory elements required for retroviral function, including promoter and enhancer regions that drive transcription of the integrated proviral genome. The LTRs play a central role in mediating reverse transcription, integration, and transcriptional activation, thereby contributing to the efficient replication and stable expression of the lentiviral vector. Their inclusion within both modified wild-type and cargo constructs facilitates vector mobilization, genomic integration, and sustained transgene expression.

[0328] In some embodiments, the 5' and 3' LTRs flank a nucleocapsid protein packaging signal, a heterologous nucleic acid insert, and minimal intervening viral sequences, enabling efficient vector assembly and transgene delivery. In certain embodiments, the modified wild-type FIV-based construct further comprises selected FIV gag sequences encoding the structural shell (capsid) proteins required for proper particle formation, in combination with other essential structural and enzymatic elements of the vector system. In some embodiments, the 5' and 3' LTRs flank a nucleocapsid protein packaging signal, a heterologous nucleic acid insert, and minimal intervening viral sequences, enabling efficient vector assembly and transgene delivery. In certain embodiments, the modified wild-type FIV-based construct further comprises selected FIV gag sequences encoding the structural shell (capsid) proteins required for proper particle formation, in combination with other essential structural and enzymatic elements of the vector system. In some embodiments, transgenes encode viral genes, including lentiviral sequences, and are designed to produce packageable vector RNA suitable for incorporation into lentiviral particles. In some embodiments, the packaging sequences include a packaging signal (SEQ ID NO: 91) and a Rev -Responsive Element (RRE) (SEQ ID NO: 293). In some embodiments, the sequence comprises the first 311Inventor: Laura A. Prendergast base pairs of the gag coding region (SEQ ID NO: 308), thereby facilitating efficient encapsidation of vector RNA within FIV capsids. It will be appreciated that inclusion of these structural, enzymatic, and regulatory sequences in the modified wild-type FIV-based construct is necessary to achieve efficient packaging, stability, and delivery of the therapeutic payload.

[0329] In various embodiments, the modified wild-type lentiviral vector construct comprises a primer binding site, SEQ ID NO: 90, and central poly-purine tract (cPPT) SEQ ID NO: 312, both of which are readily identified by one of skill in the art. These sequences are included in the vector construct, enabling the lentiviral vector to be biologically active and replication competent.

[0330] In some embodiments, the tRNA binding site SEQ ID NO: 90 is positioned just downstream of the 5' LTR SEQ ID NO: 88. In various embodiments, the tRNA binding site base-pairs with host tRNALysvia Watson-Crick interactions, serving as a primer for reverse transcriptase to initiate first-strand DNA synthesis during reverse transcription.

[0331] In various embodiments, the origin of second-strand DNA synthesis — referred to as the central polypurine tract (cPPT); SEQ ID NO: 312 — is positioned upstream of the 3' LTR SEQ ID NO: 89 within the lentiviral cargo construct. In various embodiments, the cPPT sequence initiates second-strand DNA synthesis during reverse transcription of the retroviral genome.

[0332] In various embodiments, transgenes of the present disclosure comprise 2STOP. In various embodiments, the sequence of 2STOP is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents thereof, which accomplish the dual function of trapping HIV virions, thereby preventing them from budding out of the cell membrane; and re-establishing autoinhibition over the vector’s replication once the HIV virions have been eliminated.

[0333] In various embodiments in the lentiviral cargo construct, the 2STOP dual-function protein is configured in two different ways, designated 2STOPA or 2STOPB. 2STOPA is comprised of the transcriptional repressor domain 5’ to the dominant negative domain.2STOPB is comprised of the dominant negative domain 5’ to the transcriptional repressor domain.Inventor: Laura A. Prendergast

[0334] In various embodiments in the lentiviral cargo construct, the nucleic acid sequence encoding the 2STOPA dual-function protein is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents thereof.

[0335] In various embodiments in the lentiviral cargo construct, the nucleic acid sequence encoding the 2STOPB dual-function product is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents thereof.

[0336] In various embodiments in the lentiviral cargo construct, the sequence encoding the 2STOP dual-function protein is placed downstream of a Transactivator Responsive Region (TAR), SEQ ID NO: 57, which is derived from HIV, and which up-regulates expression of TAR-labeled sequences by 100-fold.

[0337] In various embodiments in the lentiviral cargo construct, the dominant negative domain of the 2STOP gene is designated DN-lirl, SEQ ID NO: 45. The DN-lirl domain SEQ ID NO: 46, of the expressed 2STOP prevents mature HIV virions from budding out of the cells.

[0338] In various embodiments, the transcriptional repressor portion of the 2STOP dualfunction protein is encoded by a nucleic acid sequence selected from the group that includes SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and functional equivalents thereof. In various embodiments, these repressor domains consist of a group that includes Gal4-BD, GalR, GalS, Lad, Cep A, CytR, Mall, PurR, RafR, RbtR, ScrR, and functional equivalents.

[0339] In various embodiments, the transcriptional repressor portion of the 2STOP dualfunction protein comprises an amino acid sequence selected from the group that includes SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, and functional equivalents thereof. In various embodiments, these amino acid sequences correspond to repressor domains selected from a group that includes Gal4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR, and functional equivalents. In various embodiments, when expressed as part of the 2STOP protein, these repressor domainsInventor: Laura A. Prendergast dimerize and bind their cognate operator sequence to sterically inhibit transcription of downstream lentiviral sequences.

[0340] In various embodiments, the transcriptional repressor domain of the 2STOP dualfunction protein comprises a nuclear localization signal (NLS), which facilitates transport of the 2STOP protein into the nucleus of the host cell.

[0341] In various embodiments, the modified wild-type construct is comprised of an operator sequence inserted just downstream of the 5’ LTR, SEQ ID NO: 88, that is recognized and bound by the transcriptional repressor domain of the 2STOP dual-function protein to establish auto-inhibition of the lentiviral vector’s replication.

[0342] In various embodiments, the lentiviral cargo construct is comprised of an operator sequence inserted just downstream of the 5’ LTR, SEQ ID NO: 88, that is recognized and bound by the transcriptional repressor domain of the 2STOP dual-function protein to establish auto-inhibition of the lentiviral vector’s replication.

[0343] In various embodiments, the operator sequence selected for the modified wild-type lentiviral construct is identical to the operator sequence selected for the lentiviral cargo construct.

[0344] In various embodiments, the cognate operator sequence that is recognized and bound by the transcriptional repressor domain of the 2STOP is selected from the group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, and functional equivalents thereof.

[0345] In various embodiments, the operator is placed downstream of the 5 ’LTR SEQ ID NO: 88 to establish baseline inhibition over replication of the FIV construct until challenge with infectious HIV. Recognition and binding of the operator sequence by the transcriptional repressor domain contained within the 2STOP sterically inhibits transcription from the 5’ LTR, SEQ ID NO: 88, and inhibits replication of the FIV lentiviral vector.

[0346] In various embodiments, the lentiviral cargo construct is comprised of the coding and regulatory sequences that allow expression of the therapeutic dual-function gene (2STOP) which prevents HIV from budding out of the infected cells and enables the conditional auto-inhibition of the lentiviral vector.

[0347] In certain embodiments, the expression of the therapeutic or prophylactic transgenes is operably linked to a cell-type-specific promoter to confine transcriptional activity to predetermined target cell populations. Exemplary promoters include, without limitation, aInventor: Laura A. PrendergastCD4 promoter for selective expression in CD4+helper T lymphocytes, a CD8 promoter for expression in CD8+cytotoxic T lymphocytes, and a CD34 promoter for expression in hematopoietic stem and progenitor cells. It will be understood by those skilled in the art that alternative or additional promoters exhibiting equivalent or superior specificity, inducibility, or strength may be employed, either alone or in combination, without departing from the scope of the present disclosure.

[0348] In various embodiments, in the lentiviral cargo construct, the packaging sequence ( ), SEQ ID NO: 91, comprised of the first 311 nucleotides of the FIV gag gene SEQ ID NO: 308, is placed downstream of the cell-type specific promoter.

[0349] In various embodiments the lentiviral cargo construct comprises a nucleic acid sequence encoding a heterologous coat protein, to pseudo-type the lentiviral vector for transduction of a wider range of cell types, expanding vector tropism. In various embodiments, envelope glycoproteins from various viruses that can be used for this purpose are selected from a group that includes Vesicular Stomatitis Virus Glycoprotein G (VSV-G), Lymphocytic Choriomeningitis Virus (LCMV), Ross River Virus (RRV), Marburg Virus (MARV), Lassa Virus (LASV), Baculovirus GP64 (GP64), functional equivalents, and combinations thereof.

[0350] In various embodiments, the nucleic acid sequences of the envelope glycoproteins from various viruses that can be used for this purpose are selected from a group that includes VSV-G, SEQ ID NO: 296 LCMV, SEQ ID NO: 297, RRV, SEQ ID NO: 298, MARVGP, SEQ ID NO: 299 LASVGP, SEQ ID NO: 300, GP64, SEQ ID NO: 301, functional equivalents, and combinations thereof.

[0351] In various embodiments the lentiviral cargo construct comprises a constitutive internal promoter which is positioned 5’ to the nucleic acid sequence encoding the 2STOP dual-function protein, for continuous expression of the 2STOP gene.

[0352] In various embodiments, the constitutive promoter in the lentiviral cargo construct is selected from a list that includes hCMV, EF-1 alpha, CAGG and SV40 and functional equivalents thereof. In some embodiments, the constitutive promoter sequence is selected from the group consisting of hCMV SEQ ID NO: 81, EF-lalpha SEQ ID NO: 82, CAGG SEQ ID NO: 83, and SV40 SEQ ID NO: 84, and functional equivalents thereof.

[0353] In various embodiments, expression of the lentivector’s transgenes is operably linked to a cell-type-specific promoter, wherein the cell-type-specific promoter in the lentiviral cargo construct is selected from a list that includes CD4-specific promoters SEQ ID NO: 85,Inventor: Laura A. PrendergastCD8-specific promoters SEQ ID NO: 86, CD34-specific promoters SEQ ID NO:87, and functional equivalents thereof. In some embodiments, the cell-type-specific promoter sequence is configured to preferentially drive transgene expression in the corresponding target cell populations.

[0354] In various embodiments in the lentiviral cargo construct, the sequences for the promoter, or promoter / enhancer elements can be taken from expression systems such as might be readily commercially available from biotech supply companies.

[0355] In various embodiments, the lentiviral vector cargo construct for use herein additionally comprises one or more adjunctive protective transgenes configured to provide a multi-mechanistic therapeutic approach to eradicating infectious HIV from host cells. In various embodiments, such adjunctive protective transgenes include CRISPR-based gene editing, short-hairpin RNA (shRNA), nucleic acid aptamers or protein aptamers targeting HIV proteins, immunologic agents targeting HIV virions, anti -idiotypic antibodies to elicit an anti-HIV antibody response, functional equivalents, and combinations thereof.

[0356] In various embodiments, the lentiviral vector described herein comprises auxiliary transgenes targeting circulating anti-HIV antibodies in the recipient and enabling seroreconversion. Auxiliary transgenes can be selected from a list that includes, without limitation: nucleic acid aptamers and protein aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, or camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents, and combinations thereof. Each of these approaches is discussed in more detail below.

[0357] In various embodiments in the lentiviral cargo construct comprises a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system comprising a CRISPR- associated endonuclease (Cas) and signal guide ribonucleic acids (sgRNA), wherein the sgRNA is at least 15 contiguous base pairs complementary to 15 base pairs of a safe harbor site (SHS).

[0358] In various embodiments the lentiviral cargo construct comprises smaller Cas orthologues for efficient packaging into the lentiviral vector cargo construct. These smaller Cas orthologs are selected from the group consisting of Staphylococcus aureus-derived saCas9 (3.16 kb) SEQ ID NO: 96, Campylobacter jejuni-derived cjCas9 (2.95 kb) SEQ IDInventor: Laura A. PrendergastNO: 97, Staphylococcus agnetis-derived sagCas9 (2.98 kb) SEQ ID NO: 98, Cas , SEQ ID NO: 99, and functional equivalents thereof.

[0359] In various embodiments in the lentiviral cargo construct comprises signal guide RNA (sgRNA) for CRISPR-based targeted integration of the lentiviral vector construct into safe harbor loci the subject’s genome. In various embodiments, sgRNA sequences directing insertion of FIV constructs into SHSs are selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108 and the like.

[0360] In various embodiments the lentiviral cargo construct comprises sgRNA to target integration of the vector genetics into sequences coding for HIV gag-pol, integrase, env, vif, vpr, rev, vpx, and the like.

[0361] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s gag-pol genes are selected from the group consisting of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ IDNO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128 and the like.

[0362] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s integrase gene are selected from the group consisting of SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138 and the like.

[0363] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s env gene are selected from the group consisting of SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150 and the like.

[0364] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s vif gene are selected from the group consisting of SEQ ID NO: 151, SEQ ID NO: 152, SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159 and the like.

[0365] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s vpu gene are selected from the group consisting of SEQ ID NO: 160, SEQ ID NO:Inventor: Laura A. Prendergast161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168 and the like.

[0366] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s vpr gene are selected from the group consisting of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178 and the like.

[0367] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s rev gene are selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO: 190, SEQ ID NO: 191, SEQ ID NO: 192, and the like.

[0368] In various embodiments, the sgRNA sequences directing CRISPR-based knockout of HIV’s vpx gene are selected from the group consisting of SEQ ID NO: 193, SEQ ID NO: 194, and the like.

[0369] In various embodiments, the lentiviral cargo construct comprises sgRNA sequences for insertion into the nucleic acid sequence coding for the human CCR5 receptor to accomplish gene knockout. In various embodiments, the sgRNA sequences targeting the CCR5 coding sequence are selected from a list that includes SEQ ID NO: 195, SEQ ID NO:196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO:201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO:206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, and the like.

[0370] In various embodiments, the lentiviral cargo construct comprises sgRNA sequences that direct CRISPR-based excision of HIV sequences from the genome of infected host cells thereby eliminating the latent reservoir of HIV-infected cells.

[0371] In various embodiments the lentiviral cargo construct comprises genetic material that codes for short-hairpin RNA (shRNA). shRNA sequences are processed by the Dicer enzyme - which is endogenous in humans - into small interfering RNAs (siRNA) for knockdown or knockout of HIVs coding genes.

[0372] In certain embodiments, persons of skill in the art may design short hairpin RNA (shRNA) sequences using publicly available or commercially accessible online tools configured to identify optimal target sites within a given messenger RNA (mRNA) transcript while minimizing off-target effects. Non-limiting examples include the BLOCK iT™ RNAi Designer (ThermoFisher Scientific), siDESIGN Center (Horizon Discovery), siRNA WizardInventor: Laura A. Prendergast(InvivoGen), and SplashRNA platform (Broad Institute), each providing algorithm -driven prediction models and optimization features for sequence potency, specificity, and structural parameters. These tools may be used individually or in combination to facilitate the rational design of shRNA constructs for experimental or therapeutic applications.

[0373] In various embodiments the lentiviral cargo construct comprises transgenes that comprise shRNA for knockout of expressed HIV gene products. Representative HIV-1 coding sequences amenable to shRNA-mediated silencing include gag-pol, which encodes structural proteins MA, CA, NC, and p6, as well as enzymatic components PR, RT, RNase H, and IN; env. encoding the envelope glycoproteins gpl20 and gp41; and regulatory genes such as tat and rev. Accessory genes that may be targeted include, for example, nef vif, \pr. and vpu. In certain embodiments, target sequences may also be derived from HIV-2 or related lentiviruses, including vpx.

[0374] In various embodiments, the lentiviral cargo construct comprises transgenes for immunologic approaches to targeting HIV virions, including monoclonal antibodies (mAbs), broadly neutralizing monoclonal antibodies (bNmAbs), monovalent antibodies, antigen-binding fragments (Fabs), camelid nanobodies, bi-specific or multi-specific antibodies, fusion antibodies, single-chain variable fragments (scFvs), chimeric antigen receptors (CARs), immune checkpoint modulators, Fc-engineered antibodies, functional equivalents thereof, and combinations thereof.

[0375] In various embodiments the lentiviral cargo construct comprises transgenes that encode nucleic acid aptamers targeting HIV integrase, nucleocapsid, gpl20, and rev, and the like. Nucleic aptamers are short stretches of nucleic acids that bind to their targets with high affinity and specificity. Aptamers are identified using a repetitive in vitro selection and partitioning technology called SELEX (Systematic Evolution of Ligands by Exponential enrichment). The SELEX process for identification of aptamers targeting cell-surface proteins is reviewed in Pooja Dua, Soyoun Kim, Dong-ki Lee, “ Nucleic acid aptamers targeting cell-surface proteins I" Methods, Volume 54, Issue 2, 2011, pp 215-225. The existing nucleic-acid based approaches for targeting HIV gene products and the development of aptamers against HIV to inhibit virus replication, are summarized in Joshi PJ, Fisher TS, Prasad VR. Anti-HIV inhibitors based on nucleic acids: emergence of aptamers as potent antivirals. Curr Drug Targets Infect Disord. 2003 Dec;3(4):383-400.

[0376] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding protein aptamers engineered to bind HIV-related targets with high specificity andInventor: Laura A. Prendergast affinity. Owing to their reduced molecular size, capacity for efficient intracellular expression, and ability to engage otherwise inaccessible protein-protein interfaces, such aptamers are well suited for incorporation into antiviral gene therapy vectors. In certain embodiments, aptamer sequences are selected or optimized using combinatorial display technologies, including yeast, phage, or ribosome display, to achieve the desired binding characteristics.

[0377] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding anti -idiotypic antibodies configured to elicit a host-derived anti-HIV antibody response. Such anti-idiotypic antibodies mimic the antigenic determinants of HIV virions, thereby stimulating the immune system to generate antibodies that recognize and neutralize HIV. In certain embodiments, sequences encoding the anti -idiotypic antibodies are incorporated into the vector to enable sustained in vivo expression and continuous immunologic priming against HIV infection.

[0378] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding soluble decoy antigens designed to competitively bind HIV virions or HIV-specific host receptors, thereby blocking viral attachment and entry into target cells. Such decoy antigens can be derived from native HIV envelope glycoprotein domains, engineered receptor-mimetic sequences, or engineered receptor domains, and are incorporated into the vector to enable sustained in vivo production for continuous competitive inhibition of viral infection.

[0379] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding fusion proteins that incorporate antibody -derived binding domains specific for HIV virions or HIV-encoded proteins, operably linked to clearance or degradation tags to facilitate removal of the bound target from circulation or promote its intracellular degradation. Such fusion proteins may, for example, couple a single-chain variable fragment (scFv) or other antibody fragment to an Fc-engineered domain, proteasomal targeting sequence, lysosomal targeting signal, or other degradation motif. Incorporation of these constructs into the vector enables sustained in vivo production of targeted binding molecules with enhanced viral clearance capacity and potential to reduce viral load through accelerated removal or inactivation of HIV components.

[0380] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding multivalent scaffolds engineered to present multiple antibody -binding domains for enhanced capture and sequestration of HIV virions or HIV-derived antigens. Such scaffolds may be based on synthetic protein frameworks, oligomerized immunoglobulin domains, orInventor: Laura A. Prendergast self-assembling multimeric structures that increase the local density of binding sites and improve avidity for the target. Incorporation of these constructs into the vector enables sustained in vivo production of high-avidity binding platforms, thereby potentiating immune complex formation and promoting more efficient neutralization and clearance of HIV.HUMAN ANTI-RETROVIRAL DEFENSE FACTORS

[0381] In some embodiments, the lentiviral vector cargo construct comprises transgenes encoding one or more human anti-retroviral host defense factors to inhibit HIV replication and spread. Exemplary non-limiting host defense factors include apolipoprotein B mRNA-editing catalytic polypeptide-like 3G (APOBEC3G), sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), and tetherin (BST-2). These endogenous restriction factors act at distinct stages of the viral life cycle, including reverse transcription, nucleotide pool regulation, and virion release, thereby exerting complementary antiviral effects. It will be appreciated by those skilled in the art that additional host-encoded restriction factors with anti-retroviral activity are known or may be discovered, and such factors may likewise be incorporated into the vector construct, alone or in combination, without departing from the scope of the present disclosure.

[0382] In various embodiments, APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G) is an endogenous human anti-retroviral restriction factor and was the first such gene identified as an inhibitor of HIV-1 infection (Sheehy, A. M., Gaddis, N. C., Choi, J. D., & Malim, M. H. (2002). Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral vif protein. 4 / 8( August), 4-8). HIV mutants lacking a functional vif gene fail to replicate in primary CD4+T cells or macrophages. APOBEC3G induces lethal hypermutations in the retroviral genome, whereas wild-type vif protein counteracts this effect. In various embodiments, therapeutic strategies target vif to restore APOBEC3G function, for example, by delivering sgRNA sequences for CRISPR-based insertional mutagenesis or by including shRNA sequences targeting vif within the vector cargo.

[0383] In various embodiments, SAMHD1 is an intrinsic cellular enzyme that restricts HIV-1 replication in myeloid cells and quiescent CD4+T cells by depleting intracellular deoxynucleoside triphosphates (dNTPs) through its triphosphohydrolase activity, thereby blocking reverse transcription. SAMHD1 also exhibits nuclease activity against single-stranded DNA, RNA, and RNA in DNA / RNA hybrids. HIV-2 vpx promotes SAMHD1 degradation, enabling viral replication (Ballana, E., If, T. D., Este, J. A., & If, T.Inventor: Laura A. PrendergastD. (2015). SAMHD1: At the Crossroads of Cell Proliferation, Immune Responses, and Virus Restriction. Trends in Microbiology, 1-13). In various embodiments, therapeutic strategies target vpx to preserve SAMHD1 function, for example, by including sgRNA sequences in the lentivector cargo for CRISPR-mediated disruption of integrated vpx sequences, or by incorporating shRNA sequences targeting vpx within the vector cargo.

[0384] In various embodiments, human cells possess interferon-a-induced antiviral proteins, termed tetherins, which inhibit the release of mature HIV-1 virions by trapping them at the cell surface. The HIV-1 accessory protein vpu antagonizes this restriction, enabling virion release (Neil, S. J. D., Zang, T., & Bieniasz, P. D. (2008). Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu. 451 January) In various embodiments, therapeutic approaches target vpu to restore tetherin-mediated antiviral activity, for example, by including sgRNA sequences in the lentivector cargo for CRISPR-based disruption of integrated vpu sequences, or by incorporating shRNA sequences targeting vpu within the vector cargo.REGULATION OF VECTOR REPLICATION

[0385] Constructs of the present disclosure are utilized to transduce host cells. In some embodiments, the packageable vector RNA of the present disclosure is packaged into capsids. The capsid may be comprised of heterologous proteins for pseudo-typing the lentiviral vector, to aid in the delivery of the packageable vector RNA and expand the range of host cells that might be transduced.

[0386] In certain embodiments, the packageable vector RNA comprises a 5’ LTR and a 3’ LTR. The LTR sequences function in concert with reverse transcription and integration machinery to mediate stable insertion of a DNA copy of the vector genome into the host cell’s chromosomal DNA. Following integration, the vector genome is transcribed by the host cell’s transcriptional machinery, resulting in the production of multiple RNA copies of the vector, which may serve as templates for protein expression or as genomic RNA for packaging into newly formed viral particles. In some embodiments, the 5’ LTR acts as an RNA polymerase II promoter upon integration into the host cell genome.

[0387] In some embodiments, the 5’ LTR, SEQ ID NO: 88, and the 3’ LTR, SEQ ID NO: 89, of the present disclosure are lentiviral LTRs. Lentiviruses infect and integrate their genomes into dividing and non-dividing cells (e.g., neurons). In various preferred embodiments described herein, the lentiviral vector is primarily a feline immunodeficiency virus (FIV). In some embodiments, lentiviral LTR sequences are derived from FIV (i.e., shareInventor: Laura A. Prendergast at least 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% nucleic acid sequence identity with an FIV LTR).

[0388] In some embodiments, the 5’ LTR is fused with the promoter operably linked to the transgene. The 3’ LTR terminates transcription by adding a poly-A sequence at the 3’ end of the transcribed sequence.

[0389] In various embodiments, the integrase substrate element (ISE) comprises a specific nucleic acid sequence located at the termini of the reverse-transcribed viral DNA, which is recognized and bound by the viral integrase protein. The ISE contains conserved attachment sites required for proper processing and strand transfer, thereby enabling site-specific integration of the vector genome into the host cell DNA.

[0390] In various embodiments, the 5' LTR and 3' LTR of the present disclosure flank: a nucleocapsid protein packaging target site ( ), SEQ ID NO: 91, a heterologous nucleic acid insert, and minimal intervening viral sequences. The nucleocapsid protein packaging target site, also referred to as “packaging sequence,” forms secondary structures (e.g., stem -loop, bulges) that are recognized and bound by viral packaging proteins. Persons of skill in the art understand there may be intervening sequences between the transgenes carried by the lentivector.

[0391] In various embodiments, self-inactivating lentiviral vectors are engineered to integrate their therapeutic cargo into the host genome while lacking the capacity for further propagation beyond the initial transduction event. Such auto-inhibiting designs reduce the risk of excessive vector replication and minimize immune system overstimulation. In certain embodiments, it is desirable that the delivery system provides durable protection by enabling transduction of additional cell populations over time, and that expression of the therapeutic genes is inducible — activated only in response to a relevant trigger, such as exposure to infectious HIV.

[0392] In various embodiments described herein, the vector site-specifically introduces its cargo, including the therapeutic gene (2STOP), which is expressed by means of a constitutive internal promoter.

[0393] In various embodiments, the therapeutic gene product (2STOP) establishes inhibition over the replication of the delivery vector itself by means of the transcriptional repressor domain within the 2STOP, which dimerizes and binds the operator sequence engineered into the modified wild-type and cargo FIV constructs.Inventor: Laura A. Prendergast

[0394] In various embodiments, the coding sequence for 2STOP is preceded by a TAR sequence, SEQ ID NO: 57, which is recognized and bound by HIV’s transactivator protein (tat) which is expressed early after integration of the proviral HIV genome into the host DNA.by infectious HIV. Tat stabilizes TAR-labeled transcripts, upregulating expression of the 2STOP by about 100-fold. Importantly, 2STOP also comprises a dominant negative domain (DN-lirl) SEQ ID NO: 46, which traps budding HIV virions in the membrane of the infected cell.

[0395] In various embodiments, during HIV infection, the presence of numerous immature HIV particles budding from the infected cell membrane recruits the therapeutic gene product (2STOP) away from the operator sequence that negatively regulates expression of vector- encoded sequences, thereby transiently allowing vector replication. In various embodiments, upon elimination of trapped HIV virions by endogenous enzymatic digestive processes, the remaining therapeutic protein (2STOP) is liberated, allowing it to re-locate to the nucleus via the internal nuclear-localization sequence (NLS) contained within the transcriptional repressor domain of the 2STOP, whereby inhibition of vector replication is re-established.

[0396] In various embodiments, to provide sustained therapeutic activity following a single administration of the vector, the replication-repression mechanism is configured to exhibit a controlled degree of leakiness. Such leakiness permits limited, sub-threshold propagation of the vector, thereby facilitating continued transduction of additional target cell populations with the therapeutic transgenes.ALTERNATIVE APPLICATIONS FOR THE LENTIVIRAL VECTOR

[0397] Regarding the adaptation of the FIV-based vector for treatment of HIV, described in various embodiments herein, it is understood by persons of skill in the art that HIV exists in several different strains. For example, HIV-1 and HIV-2 differ in sequence by more than 55%. Numerous sub-types exist that differ from each other by 25 - 35%.

[0398] In various embodiments, this vector can be adapted for treatment of different strains of HIV and other viral infections in addition to HIV. In some embodiments, this vector can be used to deliver therapeutic genetic material for the treatment of other diseases, such as cancer, metabolic diseases, genetic disorders, other infectious diseases, or for tissue repair.

[0399] In various embodiments, the vector of the present disclosure is further suitable for ex vivo applications, wherein target cells are genetically modified outside the body and subsequently re-introduced into the subject. Exemplary uses include, without limitation, stable genetic modification of hematopoietic stem cells; introduction of therapeutic genes intoInventor: Laura A. Prendergast autologous T lymphocytes, B lymphocytes, or natural killer (NK) cells; engineering of dendritic cells for enhanced antigen presentation; and delivery of gene-silencing agents, such as short hairpin RNA (shRNA) or CRISPR-based inhibitory constructs, to selectively suppress expression of pathogenic genes.

[0398] In various embodiments the lentiviral vector described herein is useful for the eradication of HIV. The inventor recognizes that similar vectors also can be developed as a “platform technology”; i.e., a delivery system for genetic therapies that might be applied to treat other diseases. In various embodiments, the delivery mechanism of the lentiviral vector described herein can be adapted for the treatment of other viral infections. A lentiviral vector, such as the one described herein, for treatment of other viral infections comprises a dualfunction protein expressed from an internal promoter and comprised of a domain for inhibiting or obstructing an element of the target pathology and a DNA-binding transcriptional repressor domain for inhibition of the delivery vector’s own replication. In some embodiments, an operator sequence is engineered into the backbone of the delivery vector’s genetics in relationship to the promoter that drives the vector’s replication. In various embodiments, the vector’s replication is thereby negatively regulated when the operator sequence is bound by the transcriptional repressor domain contained within the therapeutic gene product. Using this approach provides, , in various embodiments, for the auto-inhibition of the vector being conditioned on the eradication of the target pathology.

[0400] With these two requirements, any replication-competent viral-based vector might be adapted for gene delivery. The dual-function protein and the operator sequences are small enough to allow additional therapeutic genes to be included in the vector cargo, even in viralbased vectors with a relatively small genome size. Other viruses that might be adapted for use as gene delivery vectors include, for example, retroviruses, lentiviruses ( Vs), adenoviruses (Ads), adeno-associated viruses (AAVs) and the like.

[0401] For a review of genetic approaches to combating hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, see Von Weizsacker, F., Wieland, S., Kock, J., Offensperger, W. B., Offensperger, S., Moradpour, D., & Blum, H. E. (1997). Gene therapy for chronic viral hepatitis: Ribozymes, antisense oligonucleotides, and dominant negative mutants. Hepatology, 26(2), 251-255.

[0402] The use of a dominant negative mutant of RAS (RASN17), and a dominant negative mutant of AKT were found to have antiproliferative, proapoptotic, and anticancer effects, representing a potential therapeutic target for pancreatic cancer (See e.g., : Stoll V, Calleja V,Inventor: Laura A. PrendergastVassaux G, Downward J, Lemoine NR. Dominant negative inhibitors of signaling through the phosphoinositol 3-kinase pathway for gene therapy of pancreatic cancer. Gut. 2005 Jan;54(l): 109-16).

[0403] The adjunctive protective transgenes described herein exhibit high target specificity, consistent with the characteristics of immunologic-based strategies for HIV intervention. Given their precise mechanism of action, it will be essential to identify the specific HIV strain infecting the subject in order to ensure optimal compatibility and therapeutic efficacy.

[0404] In certain embodiments, the lentiviral-based gene delivery system of the present disclosure is administered as an adjuvant therapy in combination with one or more established or investigational HIV treatments. In some embodiments, the vector may be co-administered with monoclonal antibodies — including broadly neutralizing antibodies (bNAbs) directed against HIV envelope glycoproteins — to enhance viral suppression and delay or prevent rebound viremia. In other embodiments, the lentivector is delivered in conjunction with antiretroviral therapy (ART), such as reverse transcriptase inhibitors, protease inhibitors, integrase strand transfer inhibitors, or entry inhibitors, to augment suppression of viral replication and target latently infected cells.

[0405] In various embodiments, the system may also be used alongside gene-editing therapies (e.g., CRISPR / Cas-based excision of integrated proviral DNA, zinc-finger nucleases, TALENs), therapeutic vaccines designed to elicit robust HIV-specific cellular immunity, or immune-modulatory agents such as checkpoint inhibitors, toll-like receptor (TLR) agonists, or cytokines. In various embodiments, the lentivector can provide durable, cell-intrinsic antiviral activity that complements the mechanism of action of other therapies as an adjuvant, thereby improving the likelihood of achieving a functional or sterilizing cure.NUCLEIC ACIDS

[0406] In some aspects, the present disclosure provides isolated nucleic acids. The isolated nucleic acids comprise a heterologous nucleic acid insert flanked by LTRs, wherein between the first LTR and the second LTR are present packaging sequences, nuclear export sequences, and minimal intervening viral sequences. In some embodiments, the first LTR is the 5’ LTR and the second LTR is the 3’ LTR.

[0407] In some aspects, the present disclosure provides transcribed nucleic acids. In some embodiments, a transcribed nucleic acid is produced in a host cell.

[0408] In some embodiments, a nuclear export sequence comprises the Rev Response Element (RRE) SEQ ID NO: 277. In some embodiments, the RRE is located between theInventor: Laura A. Prendergast sequence SEQ ID NO: 91 and the polypurine tract sequence SEQ ID NO: 278. In some embodiments, the RRE SEQ ID NO: 277 is located upstream of the sequence SEQ ID NO: 91 and the polypurine tract sequence SEQ ID NO: 278. In some embodiments, the RRE SEQ ID NO: 277 is located downstream of the sequence SEQ ID NO: 91 and the polypurine tract sequence SEQ ID NO: 312.

[0409] In some embodiments, the nucleic acid comprises minimal intervening viral sequences. In some embodiments, the minimal intervening viral sequences are up to a total of 350 base pairs in length. In some embodiments, the minimal intervening viral sequences are a total of 25-350 base pairs, 50-300 base pairs, 100-350 base pairs, 125-200 base pairs, or 10- 250 base pairs in length.

[0410] In some embodiments, the nucleic acid packageable size that will fit into the FIV lentivector is 9,400 bases.HETEROLOGOUS NUCLEIC ACID INSERTS

[0411] The disclosure relates, in various embodiments, to constructs having a heterologous nucleic acid insert carrying therapeutic genes for trapping HIV virions in infected cells and suppressing replication of the delivery vector when it is not necessary for controlling HIV infection. Such constructs, include, for example, 2STOP.

[0412] In some embodiments, the heterologous nucleic acid insert encodes regulatory sequences operably linked to expression of transgenes encoded by the vector construct.

[0413] In some embodiments, the regulatory sequences are cell-type specific promoters for expression of transgenes in specific cell types. In some embodiments, the regulatory sequences are constitutive internal promoters for continuous expression of vector-encoded transgenes.

[0414] In some embodiments the regulatory sequence is an HIV transactivator-responsive region (TAR region) that stabilizes transcripts preceded by the TAR region when recognized and bound by the HIV transactivator protein (tat), leading to 100-fold up-regulation of expression of those transcripts.

[0415] In some embodiments, the heterologous nucleic acid insert is configured to express one or more gene editing proteins in a recipient’s cells. In some embodiments, the heterologous nucleic acid insert encodes a CRISPR-associated endonuclease (Cas). In some embodiments, other gene editing machinery, such as that used for ZFN or TALEN gene editing, is incorporated in vectors described herein and encoded by a heterologous nuclei acid insert as described herein.Inventor: Laura A. Prendergast

[0416] In some embodiments, the heterologous nucleic acid insert encodes signal guide RNA (sgRNA) for targeting of CRISPR-based gene-editing approaches. sgRNA for gene editing may be readily designed by a person of skill in the art by means of design tools reviewed in Wang, J., Zhang, X., Cheng, L., & Luo, Y. (2020). An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools. RNA Biology, 17(1), 13- 22.

[0417] In some embodiments, the heterologous nucleic acid insert encodes signal guide RNA (sgRNA) for site-specific integration of the lentivector constructs into safe harbor loci in the recipient’s genome. In various embodiments, sgRNA sequences directing insertion of FIV constructs into safe harbor loci are selected from a group consisting of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and the like.

[0418] In some embodiments, the heterologous nucleic acid insert encodes signal guide RNA (sgRNA) for site-specific integration of the lentivector constructs into HIV coding sequences to accomplish gene knockout. In various embodiments, the HIV coding sequences targeted for gene knockout are selected from a group that includes gag-pol, integrase, env, vi vpu, vpr, rev, vpx, or combinations thereof.

[0419] In some embodiments, the heterologous nucleic acid insert encodes signal guide RNA (sgRNA) for site-specific integration of the lentivector constructs into the human CCR5 T-cell receptor, to knock out HIV’s preferred route into an infected person’s T-cells. sgRNA for targeted insertion of FIV constructs into the CCR5 coding region are selected the group consisting of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209 and the like.

[0420] In some embodiments, the heterologous nucleic acid insert encodes signal guide RNA (sgRNA) that directs CRISPR-based excision of HIV sequences from an infected person’s genome.

[0421] In some embodiments, the heterologous nucleic acid inserts encode adjunctive protective transgenes for attacking HIV in an infected person. In some embodiments, these adjunctive protective transgenes comprise immunologic agents for targeting HIV virions in the infected host. Wherein the immuno-based approaches for targeting HIV are selected from a list that includes monoclonal antibodies (mAbs), monovalent antibodies (mAbs),Inventor: Laura A. Prendergast antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bi specific antibodies, engineered immuno-adhesins, functional equivalents and combinations thereof.

[0422] In some embodiments, the adjunctive protective transgenes comprise short hairpin RNA (shRNA) for knockout of expressed HIV gene products. Exemplary target genes for shRNA-mediated silencing of HIV- 1 coding sequences include, for example, gag-pol (encoding structural precursors MA, CA, NC, p6, and enzymatic components PR, RT, RNase H, and IN), env (encoding envelope glycoproteins gpl20 and gp41), and regulatory genes such as tat and rev. Accessory genes include nef vif, vpr, and vpu. In some embodiments, sequences derived from HIV-2 or related lentiviruses, such as vpx, may also be targeted or incorporated.

[0423] In some embodiments, the adjunctive protective transgenes comprise nucleic acid aptamers or protein aptamers targeting expressed HIV gene products. Exemplary target genes for aptamer- or small-peptide-based targeting of HIV- 1 coding sequences include gag-pol (encoding structural precursors MA, CA, NC, p6, and enzymatic components PR, RT, RNase H, and IN), env (encoding envelope glycoproteins gpl20 and gp41), and regulatory genes such as tat and rev. Accessory genes include nef, vif vpr, and vpu. In some embodiments, sequences derived from HIV-2 or related lentiviruses, such as vpx, can also be targeted or incorporated.

[0424] In some embodiments, the heterologous nucleic acid insert encodes auxiliary transgenes comprising molecular strategies for binding, neutralizing, or otherwise modulating circulating anti-HIV antibodies to accomplish seroreconversion.

[0425] In some embodiments, the heterologous nucleic acid insert encodes heterologous envelope glycoproteins for pseudotyping the vector to broaden the lentiviral vector’s tropism for host cells.ALTERNATIVE ENVELOPE PROTEINS FOR EXPANDING HOST TROPISM

[0426] In various embodiments, the vector is pseudotyped with an alternative envelope protein derived from viruses such as Vesicular Stomatitis Virus Glycoprotein G (VSV-G), Lymphocytic Choriomeningitis Virus (LCMV), Ross River Virus (RRV), Marburg Virus (MARV GP), Lassa Virus (LASV GP), Baculovirus GP64 (GP64), functional equivalents, and combinations thereof.

[0427] In some embodiments, the nucleic acid sequence encoding the heterologous coat protein is selected from a group consisting of VSV-G, SEQ ID NO: 296 LCMV, SEQ ID NO:Inventor: Laura A. Prendergast297, RRV, SEQ ID NO: 298, MARVGP, SEQ ID NO: 299 LASVGP, SEQ ID NO: 300, GP64, SEQ ID NO: 301, functional equivalents and combinations thereof. Selection of these envelope proteins enables modulation of vector tropism, allowing targeted delivery to specific cell types or tissues relevant to HIV infection or other viral pathologies.

[0428] In certain embodiments, the vector is pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G) SEQ ID NO: 296, which confers broad tropism enabling transduction of a wide range of mammalian cell types, including hematopoietic, neuronal, glial, endothelial, epithelial, muscular, and hepatic cells. VSV-G-pseudotyped vectors efficiently transduce both dividing and non-dividing cells through interaction with widely expressed LDL receptor family members.

[0429] In certain embodiments, the vector is pseudotyped with the lymphocytic choriomeningitis virus glycoprotein (LCMV-GP) SEQ ID NO: 297, which confers efficient transduction of neuronal, hepatocytic, dendritic, and other immune-derived cells.LCMV-GP-pseudotyped vectors can cross the blood-brain barrier and efficiently transduce both dividing and non-dividing cells, enabling broad application in central nervous system and systemic delivery contexts.

[0430] In certain embodiments, the vector is pseudotyped with the envelope glycoprotein of Ross River virus (RRV Env), an alphavirus envelope protein, as set forth in SEQ ID NO:298. RRV Env exhibits preferential tropism for B lymphocytes and other hematopoietic lineage cells, while retaining the capacity to transduce both adherent and non-adherent cell types. Pseudotyping with RRV Env enables targeted delivery to immune cell compartments.

[0431] In certain embodiments, the vector is pseudotyped with the Marburg virus glycoprotein (MARV-GP) SEQ ID NO: 299, which mediates entry into a broad range of mammalian cells, including hepatocytes, endothelial cells, macrophages, and dendritic cells. MARV-GP pseudotyping supports systemic delivery and efficient transduction of both immune and parenchymal tissues.

[0432] In certain embodiments, the vector is pseudotyped with the Lassa virus glycoprotein (LASV-GP) SEQ ID NO: 300, which targets hepatocytes, endothelial cells, macrophages, and certain epithelial cell populations. LASV-GP-pseudotyped vectors can mediate broad systemic transduction and are compatible with both dividing and non-dividing cell targets.

[0433] In certain embodiments, the vector is pseudotyped with the baculovirus envelope glycoprotein GP64 SEQ ID NO: 301, which enables transduction of hepatocytes, epithelial cells, and certain neuronal populations, and facilitates entry into a broad range of vertebrateInventor: Laura A. Prendergast and invertebrate cells. GP64 pseudotyping is compatible with systemic or localized delivery to diverse tissue types.HOST CELLS

[0434] In various embodiments, the present disclosure provides for the use of host cells as vehicles for the delivery, expression, or maintenance of the nucleic acid constructs described herein. The host cells may be of prokaryotic or eukaryotic origin, and may be naturally occurring, genetically modified, or otherwise engineered to enhance compatibility with the vector system. In certain embodiments, the host cells are selected for their ability to stably maintain the integrated vector genome, thereby enabling long-term or inducible expression of the therapeutic transgene(s).

[0435] In some embodiments, the host cells are mammalian cells, including but not limited to primary cells, established cell lines, or progenitor cell populations. Non-limiting examples include human embryonic kidney (HEK) cells, Chinese hamster ovary (CHO) cells, primary fibroblasts, mesenchymal stem cells, hematopoietic stem cells, and induced pluripotent stem cells (iPSCs). In certain embodiments, the host cells are selected to match the recipient’s genotype (autologous cells) to minimize immune rejection, or are allogeneic cells engineered to reduce immunogenicity through expression of immune-modulatory proteins or deletion of immunologically relevant loci.

[0436] In some embodiments, the host cells are utilized in ex vivo therapeutic workflows. For example, hematopoietic stem cells may be isolated from a subject, transduced with the vector to incorporate therapeutic or regulatory genes, and subsequently re-infused into the same subject to restore or augment immune function. Similarly, autologous T lymphocytes may be engineered to express virus-specific receptors, such as chimeric antigen receptors (CARs), or to secrete broadly neutralizing antibodies, thereby conferring targeted immunity against HIV or other pathogens.

[0437] In further embodiments, host cells are employed as in vitro production platforms for vector manufacturing. In such cases, the host cells may be engineered to optimize vector yield, stability, and infectivity, and may include packaging cell lines harboring helper plasmids, inducible expression systems, or genetic safeguards to prevent replication-competent lentivirus formation. The use of such engineered host cells facilitates scalable, reproducible production of clinical -grade vector preparations in compliance with current good manufacturing practice (cGMP) requirements.METHODS OF USEInventor: Laura A. Prendergast

[0438] In some aspects, the present disclosure provides methods for introducing, or delivering, a lentiviral vector carrying therapeutic transgenes for the eradication of HIV virions from infected host cells.

[0439] In some aspects, the present disclosure provides methods for assessing the type of virus to be treated in a subject. The method comprises obtaining a biological sample from the subject, isolating viral particles or nucleic acids from the sample, and analyzing the isolated material using one or more diagnostic assays to determine the viral species, subtype, clade, or strain. The analysis may include, for example, nucleic acid sequencing, polymerase chain reaction (PCR) with virus-specific primers, hybridization assays, antigen detection, or serologic testing. The viral typing information is used to guide selection of an appropriate therapeutic lentiviral vector, envelope glycoprotein pseudo-type, cargo sequence, dosing regimen, or route of administration for treatment of the identified virus.

[0440] In various embodiments, the methods of the present disclosure comprise administering to a subject in need thereof an FIV-based lentiviral vector cargo construct as described herein. The vector may comprise, in certain embodiments, one or more heterologous expression cassettes encoding therapeutic or prophylactic transgenes directed against HIV. Such transgenes include, without limitation, CRISPR-based gene editing components, short-hairpin RNA (shRNA) sequences, protein or nucleic acid aptamers, immunologic agents, anti -idiotypic antibodies, soluble decoy antigens, fusion proteins incorporating antibody binding domains with clearance or degradation tags, multivalent scaffolds for enhanced antibody capture, human anti-retroviral host defense factors, or combinations thereof. In some embodiments, the vector is pseudotyped with a heterologous envelope glycoprotein, such as baculovirus GP64, to broaden or alter target cell tropism. The methods described herein are applicable to human subjects diagnosed with HIV infection, including acute, chronic, or latent stages of disease. In certain embodiments, the methods are employed for prophylactic administration to subjects at elevated risk of acquiring HIV infection. In further embodiments, the methods are applicable to the treatment or prevention of infection by other related lentiviruses or retroviruses in human or veterinary contexts. The subject population may include individuals who are naive to antiretroviral therapy, those currently receiving antiretroviral therapy, or those experiencing virologic failure despite treatment. In various embodiments, the FIV-based lentiviral vector cargo construct, or a nucleic acid construct encoding same, is administered in vivo directly to the subject by injection into the bloodstream or into a specific organ, or by another suitable route ofInventor: Laura A. Prendergast administration. The route of administration may be selected from the group consisting of intravenous, intra-arterial, intradermal, transdermal, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, intraperitoneal, intratumoral, perfusion, lavage, intracerebral, intrathecal, and other mucosal or localized delivery routes. In certain embodiments, the route is selected based on the HIV strain or tissue tropism of the infection.

[0441] In some embodiments, the methods comprise ex vivo modification of target cells, wherein cells are harvested from the subject, transduced with the vector under controlled culture conditions, and subsequently reintroduced into the subject. Target cells for ex vivo transduction may include, without limitation, CD4+T lymphocytes, hematopoietic stem and progenitor cells (e.g., CD34+cells), or other immune cell subsets relevant to HIV pathogenesis. In further embodiments, administration may be localized to specific anatomical sites, such as lymphoid tissues, bone marrow, or other reservoirs of HIV infection, to enhance targeting efficiency and therapeutic effect. Administration may be performed by syringe or any other method suitable for injection or delivery of a solution.

[0442] In some aspects, the present disclosure provides a method of transducing a host cell with a therapeutic transgene encoding a dual-function 2STOP protein, integrating the lentiviral vector cargo into a safe harbor site of the host cell genome, constitutively expressing the 2STOP protein from a constitutive internal promoter, and inhibiting replication of the lentiviral vector in the host cell by binding of a transcriptional repressor domain of the 2STOP protein to a cognate operator sequence located downstream of a 5' LTR of the lentiviral vector.

[0443] In some aspects, the present disclosure provides a method that further comprises, upon exposure of the host cell to HIV, up-regulating 2STOP protein expression at least 100-fold through interaction of the HIV tat protein with a TAR sequence positioned upstream of the 2STOP coding sequence. 2STOP prevents HIV virions from budding from infected cells via a dominant-negative DN-lirl domain of the 2STOP protein, routing trapped HIV particles into endogenous degradative pathways for enzymatic elimination.

[0444] In some aspects, the present disclosure provides a method that further comprises, upon challenge of the host cell with HIV, recruiting the 2STOP protein away from the operator sequence, thereby trapping HIV virions from budding from infected cells. This provides transient relief of auto-inhibition over the replication of the lentiviral vector thereby providing for transduction of additional host cells.Inventor: Laura A. Prendergast

[0445] In some aspects, the present disclosure provides a method that further comprises, upon eradication of HIV virions from the host cell, re-establishing inhibition of lentiviral vector replication by translocation of the 2STOP protein into the nucleus via a nuclear localization sequence, and binding of the transcriptional repressor domain to the cognate operator sequence, thereby re-establishing repression of the lentivector’ s replication.

[0446] A method of administering a lentiviral vector to a subject comprises formulating the lentiviral vector in a pharmaceutically acceptable carrier and delivering the formulated composition to the subject, wherein the pharmaceutically acceptable carrier comprises one or more components selected from the group consisting of solvents, dispersion media, vehicles, coatings, diluents, antibacterial agents, antifungal agents, isotonic agents, absorption-delaying agents, buffers, carrier solutions, suspensions, and colloids, and wherein the carrier is selected to avoid producing an allergic or other untoward reaction when administered to the subject.

[0447] In some aspects, the present disclosure provides methods for introducing a lentiviral vector carrying one or more therapeutic transgenes for the eradication, suppression, or functional cure of virions from a pathogenic virus infecting a host cell. The pathogenic virus may be any virus capable of infecting mammalian cells, including but not limited to retroviruses, hepadnaviruses, flaviviruses, herpesviruses, filoviruses, arenaviruses, poxviruses, orthomyxoviruses, coronaviruses, paramyxoviruses, and parvoviruses, as well as related or emerging viral pathogens.

[0448] In various embodiments, the FIV-based lentiviral vector cargo construct, or nucleic acid construct encoding same, is administered in a therapeutically effective amount sufficient to achieve transduction of the desired target cell population and to confer the intended therapeutic or prophylactic effect. In certain embodiments, the dose is expressed as a quantity of transducing units (TU) per milliliter, vector genomes (vg) per kilogram of body weight, or an equivalent measure known in the art. Exemplary non-limiting doses may range from about 105to about 1012TU / mL, or from about 106to about 1011vg / kg, administered in a single infusion or as multiple doses over a defined treatment period. In some embodiments, administration is performed as a single dose; in others, repeated dosing at predetermined intervals is employed to achieve or maintain therapeutic effect. Dose selection may be adjusted according to the route of administration, the HIV strain or clade to be targeted, the stage of infection, and the patient’s clinical condition. Dose-escalation regimens, as known inInventor: Laura A. Prendergast the art, may be used to determine the maximum tolerated or optimal biologically effective dose for a given patient population.

[0449] In certain embodiments, the methods of the present disclosure comprise administering the FIV-based lentiviral vector cargo construct, or nucleic acid construct encoding same, in combination with one or more additional therapeutic agents or interventions. Such combination therapies may include, without limitation, concurrent or sequential administration with antiretroviral therapy (ART) regimens comprising reverse transcriptase inhibitors, protease inhibitors, integrase strand transfer inhibitors, or entry inhibitors; administration with immunologic agents, including monoclonal antibodies, broadly neutralizing monoclonal antibodies, bi-specific or multi-specific antibodies, or immune checkpoint modulators; administration with host defense factor modulators; or administration with pharmacologic agents that promote latency reversal or immune clearance of HIV-infected cells. In some embodiments, combination therapy is employed to enhance the potency, breadth, and durability of the therapeutic effect, to reduce the likelihood of viral escape, and to target multiple stages of the HIV life cycle.KITS AND RELATED COMPOSITIONS

[0450] In some embodiments, the instant disclosure relates to a kit for administering a replication-competent lentiviral vector for treating, curing, or neutralizing HIV from a subject in need of treatment.

[0451] A kit capable of being assembled can comprise, for example, a vial, tube or other container, comprising lentivector constructs, enclosed in an FIV coat. The wild-type construct comprises the vector backbone, including elements essential for vector replication, and an operator sequence for auto-repression appropriate for the transcriptional repressor domain chosen for inclusion in the 2STOP protein. The cargo construct comprises the therapeutic gene (2STOP), and the cognate operator sequence necessary for the vector’s auto-regulation (identical to the cognate operator sequence on the modified wild-type strand). In various embodiments, such kits can be prepared for sale to customers, hospitals, or labs. In some embodiments, a kit capable of being assembled can comprise nucleic acid aptamers or peptide aptamers; short inhibitory peptides; immunologic agents; soluble receptor decoys; host restriction factors, including TRIM5a, APOBEC3G, or SAMHD1; fusion or entry inhibitors targeting gpl20 or gp41; vaccine-derived immunogens to elicit anti -HIV immune responses; small interfering RNA (siRNA) or short hairpin RNA (shRNA) for RNA interference-mediated gene silencing; ribozymes or antisense oligonucleotides forInventor: Laura A. Prendergast post-transcriptional suppression; and genome editing systems, including CRISPR / Cas constructs with corresponding single-guide RNAs (sgRNAs), zinc-finger nucleases (ZFNs), or transcription activator-like effector nucleases (TALENs) for targeted modification or knockout of viral or host genes essential for HIV replication, combinations thereof, and the like.

[0452] In some embodiments, a kit capable of being assembled can comprise buffers, enzymes, and reagents for cloning protective transgenes into the lentiviral vector backbone, including, without limitation: DNA and RNA primers and probes for PCR, qPCR, and RT-PCR; reverse transcriptase, high-fidelity DNA polymerases, and nucleotide substrates dNTPs, rNTPs; restriction enzymes, ligases, linkers, adaptors, and reagents for seamless cloning or assembly; nucleic acid purification and concentration kits; sequencing reagents for verification of transgene insertion and orientation; cell culture media, sera, antibiotics, and supplements for propagation of vector-bearing cells; transduction-enhancing agents such as polybrene or protamine sulfate; and quality-control reagents, standards, or markers for confirming transgene expression, genomic integration, or functional activity.

[0453] In some embodiments, a kit capable of being assembled can comprise reagents and ancillary components for performing antibody -based assays, including, without limitation, Western blot and enzyme-linked immunosorbent assay (ELISA) formats, to detect and characterize specific HIV strains present in a biological sample. Data obtained from such strain-typing assays may be utilized to inform the selection of one or more anti-HIV protective transgenes for incorporation into the vector cargo strand, thereby enabling customization of the vector backbone to target the particular HIV strain infecting the subject.

[0454] In some aspects, the present disclosure provides a kit configured for use in identifying and characterizing a target pathogen or pathological condition in a subject, and for guiding customization of a therapeutic lentiviral vector for treatment. The kit may comprise reagents for antibody -based assays, such as Western blot or ELISA, for detecting and differentiating among viral strains or subtypes, including but not limited to retroviruses, hepadnaviruses, flaviviruses, herpesviruses, filoviruses, arenaviruses, poxviruses, orthomyxoviruses, coronaviruses, paramyxoviruses, and parvoviruses. In certain embodiments, the kit further comprises reagents for detecting biomarkers associated with non-viral pathologies, such as oncologic, genetic, or autoimmune disorders, to determine suitability of the vector for delivering a corresponding therapeutic transgene. Selection of specific therapeutic strategies — including one or more transgenes, targeting domains, orInventor: Laura A. Prendergast regulatory control elements — for inclusion in the lentiviral vector cargo may be based on the identified pathogen strain, disease subtype, or pathological profile of the subject.

[0455] The kit can be designed to facilitate use of the methods described herein by researchers and can take many forms. Each of the compositions of the kit, where applicable, may be provided in liquid form (e.g., in solution), or in solid form, (e.g., a dry powder). In certain cases, some of the compositions may be constituted or otherwise processed (e.g., to an active form), for example, by the addition of a suitable solvent or other species (for example, water or a cell culture medium), which may or may not be provided with the kit.

[0456] In various embodiments, the kit can include instructions for mixing one or more components of the kit and / or isolating and mixing a sample and applying to a subject. As used herein, "instructions" can define a component of instruction and / or promotion, and typically involve written instructions on or associated with packaging of the disclosure. Instructions also can include any oral or electronic instructions provided in any manner such that a user will clearly recognize that the instructions are to be associated with the kit, for example, audiovisual (e.g., videotape, DVD, etc.), Internet, and / or web-based communications, etc. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions can also reflect approval by the agency of manufacture, use or sale for animal administration. In certain embodiments, the instructions further comprise guidelines for the design of primers, shRNA molecules, or sgRNA sequences appropriate to the strain of HIV, or other virus, to be targeted.

[0457] The kit can, in various emboidments, comprise one or more of the components described herein in one or more containers, including, without limitation, a container housing agents described herein. The agents may be in the form of a liquid, gel or solid (powder). The agents may be prepared sterilely, packaged in syringe and shipped refrigerated. Alternatively, it may be housed in a vial or other container for storage. A second container may have other agents prepared sterilely. Alternatively, the kit can comprise agents premixed and shipped in a syringe, vial, tube, or other container.

Claims

AMENDED CLAIMS received by the International Bureau on 14 FEB 2026 (14.02.2026)1. A vector for eradicating human immunodeficiency virus (HIV) from a human, comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV.

2. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 1, wherein the vector is a lentiviral vector.

3. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 2, wherein the lentiviral vector is a feline immunodeficiency virus (FlV)-based vector.

4. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 3, wherein the FIV-based vector comprises a modified wild type FIV strand and an FIV cargo strand.

5. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the FIV cargo strand comprises the nucleic acid sequence encoding the dualfunction protein which inhibits infectious HIV from budding out of HIV-infected cells and represses replication of the vector in the absence of HIV.

6. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 5, wherein the nucleic acid sequence on the FIV cargo strand that encodes the dual function protein is 2STOP.

7. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 1, wherein the dual -function protein is 2STOP.

8. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 7, wherein 2STOP comprises a transcriptional repressor domain that represses vector replication, and a dominant negative domain that inhibits budding of HIV from membranes of HIV infected cells.

9. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 8, wherein the 2STOP protein is 2STOPA and is encoded by a nucleic acid sequence designated 2STOPA and wherein the sequence encoding the transcriptional repressor domain of 2STOPA is 5’ of the sequence encoding the dominant negative domain.

10. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 9, wherein the nucleic acid sequence encoding 2STOPA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents thereof.

11. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 9, wherein the amino acid sequence of 2STOPA is selected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and functional equivalents thereof.

12. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 11, wherein the 2STOPA comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to any one of the amino acid sequences set forth in the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and functional equivalents thereof.

13. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 8, wherein the 2STOP protein is 2STOPB and is encoded by a nucleic acid sequence designated 2STOPB, and wherein the nucleic acid sequence encoding the dominant negative domain of 2STOPB is 5’ of the sequence encoding the transcriptional repressor domain.

14. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 13, wherein the nucleic acid sequence designated 2STOPB is selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, and functional equivalents thereof.

15. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 13, wherein the amino acid sequence of 2STOPB is selected from the group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and functional equivalents thereof.

16. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 15, wherein the 2STOPB comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99% amino acid sequence identity to any one of the amino acid sequences set forth in a group consisting of SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, and functional equivalents thereof.

17. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 8, wherein the domain for inhibiting budding of HIV from membranes of HIV infected cells is encoded by a sequence designated DN-lirl.

18. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 8 wherein DN-lirl is set forth in SEQ ID NO: 45.

19. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 18, wherein the domain for inhibiting budding of HIV from membranes of HIV infected cells is of DN-lirl.

20. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 19, wherein the amino acid sequence of DN-lirl is set forth in SEQ ID NO: 46.

21. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 19, wherein DN-lirl comprises an amino acid sequence having at least 85%, 90%, 96%, 97%, 98 %, or 99%, amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 46.

22. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 8, wherein the transcriptional repressor domain is selected from a group consisting of Gal4-BD, GalR, GalS, LacI, CcpA, CytR, Mall, PurR, RafR, RbtR, ScrR and functional equivalents thereof.

23. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 22, wherein the sequence encoding the transcriptional repressor domain is selected from a group consisting of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 and functional equivalents thereof.

24. A vector according to claim 22, wherein the transcriptional repressor domain sequence comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and functional equivalents thereof.

25. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 6, wherein the 2STOP coding sequence is downstream of a Transactivation Response Region (TAR sequence).

26. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 25, wherein the TAR sequence is set forth in SEQ ID NO: 69.10727. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the modified wild type FIV strand comprises an autoinhibition operator sequence downstream of the native 5’ LTR sequence as set forth in SEQ ID NO: 88.

28. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 25, wherein an operator sequence on the cargo strand is identical to the autoinhibition operator sequence on the modified wild-type strand.

29. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 28, wherein the dual-function 2STOP protein comprises a cognate transcriptional repressor that specifically recognizes the operator sequence on the modified wild-type strand and the cargo strand.

30. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 28 wherein the sequence of the operator sequence on the modified wild-type strand and on the cargo strand is selected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, and functional equivalents thereof.

31. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 5, wherein the cargo strand further comprises a constitutive internal promoter operably linked to the 2STOP coding sequence.

32. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 31, wherein the constitutive internal promoter is selected from the group consisting of hCMV, EF-1 alpha, CAGG and SV40, and functional equivalents thereof.

33. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 32, wherein the constitutive internal promoter sequence is selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, and functional equivalents thereof.

34. A lentiviral vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the replication of the lentivector is placed under the control of a cell-type specific promoter sequence.

35. The lentiviral vector of claim 34, wherein the promoter is a cell -type-specific promoter selected from the group consisting of a CD4 promoter, a CD8 promoter, a CD34 promoter, and functional equivalents thereof.10836. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the FIV wild-type strand comprises a 5’ Long Terminal Repeat (LTR), and 3’ LTR, a primer binding site (PBS), and a sequence for packaging the lentivector genomic strand into the FIV shell.

37. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 36, wherein the FIV cargo strand further comprises one or more adjunctive protective transgenes.

38. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 37, wherein the one or more adjunctive protective transgenes comprise one or more nucleic acid sequences coding for a gene-editing technology.

39. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 38, wherein the gene-editing technology is selected from Transcriptional Activator Effector Nucleases (TALENs), Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based editing, Zinc Finger Nucleases (ZFNs), or combinations thereof.

40. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 39, further comprising a CRISPR-associated (Cas) endonuclease system directed by a signal guide RNA (sgRNA).

41. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 40, wherein the CRISPR-associated endonuclease can be selected from the group consisting of Staphylococcus aureus-derived saCas9 (3.16 kb), Campylobacter jejuni -derived cjCas9 (2.95 kb), Staphylococcus agnetis-derived sagCas9 (2.98 kb), Cas , and functional equivalents thereof.

42. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 40, wherein the vector cargo is inserted into a safe harbor locus or an inessential gene, including the CCR5 chemokine receptor gene, or into HIV coding sequences for targeted HIV gene knockout to impair viral integration.

43. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 40, wherein the sgRNA sequence for insertion of the FIV-based vector constructs into genomic safe harbor loci are selected from the group consisting of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and functional equivalents.

44. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 42, wherein the sgRNA sequence for insertional of the FIV-based vector into the109CCR5 chemokine receptor gene are selected from the group consisting of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO: 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO: 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO: 208, SEQ ID NO: 209, and functional equivalents.

45. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 42, wherein the HIV coding sequences targeted for insertional knockout are selected from gag-pol, integrase, env, vif, vpu, vpr, rev, vpx, or combinations thereof.

46. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein the sgRNA sequences for insertion of the FIV-based vector genome into HIV’ s gag-pol sequence are selected from the group consisting of SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ IDNO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ IDNO: 120, SEQ ID NO: 121, SEQ ID NO: 122 SEQ ID NO: 123, SEQ ID NO: 124, SEQ IDNO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, and functional equivalents thereof.

47. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein the sgRNA sequence for insertion of the FIV-based vector genome into HIV integrase sequence to accomplish gene knockout are selected from SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and functional equivalents thereof.

48. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein the sgRNA sequence for insertion of the FIV-based vector genome into HIV env sequence are selected from SEQ ID NO: 139, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 143, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 148, SEQ ID NO: 149, SEQ ID NO: 150, and functional equivalents thereof.

49. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein the sgRNA sequences for insertion of the FIV-based vector genome into HIV vif sequence are selected from SEQ ID NO: 151, SEQ ID NO: 152, ,SEQ ID NO: 153, SEQ ID NO: 154, SEQ ID NO: 155, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, and functional equivalents thereof.

50. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein the sgRNA sequences for insertion of the FIV-based vector genome into110HIV vpu sequence are selected from the group consisting of SEQ ID NO: 160, SEQ ID NO: 161, SEQ ID NO: 162, SEQ ID NO: 163, SEQ ID NO: 164, SEQ ID NO: 165, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 168, and functional equivalents thereof.

51. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpr sequence are selected from the group consisting of SEQ ID NO: 169, SEQ ID NO: 170, SEQ ID NO: 171, SEQ ID NO: 172, SEQ ID NO: 173, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, and functional equivalents thereof.

52. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein sgRNA sequences for insertion of the FIV-based vector genome into HIV’s rev sequence are selected from the group consisting of SEQ ID NO: 179, SEQ ID NO: 180, SEQ ID NO: 181, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO:185, SEQ ID NO: 186, SEQ ID NO: 187, SEQ ID NO: 188, SEQ ID NO: 189, SEQ ID NO:190, SEQ ID NO: 191, SEQ ID NO: 192, and functional equivalents thereof.

53. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 45, wherein sgRNA sequences for insertion of the FIV-based vector genome into HIV’s vpx sequence are selected from the group consisting of SEQ ID NO: 193, SEQ ID NO: 194, and functional equivalents thereof.

54. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 54, wherein sgRNA sequences for insertion of the FIV-based vector genome into the human CCR5 T-cell receptor are selected from the group consisting of SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197, SEQ ID NO 198, SEQ ID NO: 199, SEQ ID NO: 200, SEQ ID NO: 201, SEQ ID NO: 202, SEQ ID NO 203, SEQ ID NO: 204, SEQ ID NO: 205, SEQ ID NO: 206, SEQ ID NO: 207, SEQ ID NO 208, SEQ ID NO: 209, and functional equivalents thereof.

55. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 39, wherein the CRISPR-based gene editing comprises one or more sequences that direct excision of HIV sequences from the genome of infected host cells.

56. A vector eradicating for eradicating human immunodeficiency virus (HIV) from a human according to claim 37, wherein the one or more adjunctive protective transgenes further comprise shRNA sequences for targeted knockout of HIV gene products.11157. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 56, wherein the HIV gene products selected for knockout by shRNA are selected from the group consisting of gag-pol, integrase, env, vif, vpu, vpr, rev, vpx, and the like.

58. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for gag-pol knockout are selected from the group consisting of SEQ ID NO: 210, SEQ ID NO: 211, SEQ ID NO: 212, SEQ ID NO: 213, SEQ ID NO: 214, SEQ ID NO: 215, SEQ ID NO: 216, SEQ ID NO: 217, SEQ ID NO: 218, SEQID NO: 219, SEQ ID NO: 220, SEQ ID NO: 221, SEQ ID NO: 222, SEQ ID NO: 223, SEQID NO: 224, SEQ ID NO: 225, SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231, SEQ ID NO: 232, SEQ ID NO: 233, and functional equivalents.

59. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for integrase knockout are selected from the group consisting of SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 236, SEQ ID NO: 237, SEQ ID NO: 238, and functional equivalents thereof.

60. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for env knockout are selected from the group consisting of SEQ ID NO: 239, SEQ ID NO: 240, SEQ ID NO: 241, SEQ ID NO: 242, SEQ ID NO: 243, SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, and functional equivalents thereof.

61. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for vif knockout are selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251, SEQ ID NO: 252, SEQ ID NO: 253, and functional equivalents thereof.

62. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for vpu knockout are selected from the group consisting of SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, and functional equivalents thereof.

63. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for vpr knockout are selected from the group consisting of SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261, SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265, and functional equivalents thereof.11264. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for rev knockout are selected from the group consisting of SEQ ID NO: 266, SEQ ID NO: 267, SEQ ID NO: 268, SEQ ID NO: 269, SEQID NO: 270, SEQ ID NO: 271, SEQ ID NO: 272, SEQ ID NO: 273, SEQ ID NO: 274, SEQID NO: 275, SEQ ID NO: 276, SEQ ID NO: 277, SEQ ID NO: 278, SEQ ID NO: 279, SEQID NO: 280, SEQ ID NO: 281, SEQ ID NO: 282, SEQ ID NO: 283, SEQ ID NO: 284, SEQID NO: 285, and functional equivalents thereof.

65. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 57, wherein the shRNA sequences for vpx knockout are selected from the group consisting of SEQ ID NO. 286, SEQ ID NO. 287, SEQ ID NO. 288, SEQ ID NO. 289, SEQ ID NO. 290, SEQ ID NO. 291, SEQ ID NO. 292, SEQ ID NO. 293, SEQ ID NO. 294, SEQ ID NO. 295, and functional equivalents thereof.

66. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 37, wherein the adjunctive protective transgenes encode immunologic agents for targeting HIV virions in an infected host.

67. The vector of claim 66, wherein the immunologic agent is selected from the group consisting of monoclonal antibodies (mAbs), monovalent antibodies (mAbs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bi-specific antibodies, engineered immuno-adhesins, functional equivalents, and combinations thereof.

68. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 37, wherein the adjunctive protective transgenes comprise nucleic acid aptamers, protein aptamers, functional equivalents and combinations thereof, for targeting HIV virions in an infected host.

69. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the FIV cargo strand further comprises auxiliary transgenes for targeting circulating anti-HIV antibodies in the infected host to accomplish seroreconversion.

70. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 69, wherein the auxiliary transgenes for seroreconversion are comprised of, without limitation, nucleic acid aptamers or peptide aptamers; immunologic agents; short inhibitory peptides or mimotopes; antibody fragments including Fabs, scFvs, or camelid-derived nanobodies; soluble decoy antigens or engineered receptor domains; fusion proteins113incorporating antibody -binding domains with clearance or degradation tags; multivalent scaffolds for enhanced antibody capture; functional equivalents; and combinations thereof.

71. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 4, wherein the FIV cargo strand further comprises a nucleic acid sequence encoding a heterologous envelope glycoprotein for pseudo-typing the lentivector.

72. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 71, wherein the heterologous envelope glycoprotein is taken from a virus selected from the group consisting of Vesicular Stomatitis Virus, Lymphocytic choriomeningitis virus (LCMV), Alphavirus Ross River virus (RRV), Marburg virus, Lassa virus, Baculovirus, functional equivalents, and combinations thereof.

73. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 72, wherein the nucleic acid sequences encoding the heterologous envelope protein are selected from a group consisting of INF-G, SEQ ID NO: 296, LCMV-GP, SEQ ID NO: 297, RRVE1 / E2, SEQ ID NO: 298, MARV, SEQ ID NO: 299, LASV, SEQ ID NO: 300, and GP6R SEQ ID NO: 301, combinations, and functional equivalents thereof.

74. A vector for eradicating human immunodeficiency virus (HIV) from a human according to claim 72, wherein the heterologous envelope glycoprotein has an amino acid sequence selected from the group consisting of SEQ ID NO: 302, SEQ ID NO: 303, SEQ ID NO: 304, SEQ ID NO: 305, SEQ ID NO: 306, SEQ ID NO: 307, SEQ ID NO: 308, and functional equivalents thereof.

75. A method for eradication of HIV from an HIV-infected subject, comprising delivering a vector comprising a nucleic acid sequence encoding a dual-function protein that inhibits infectious HIV from budding from HIV-infected cells and represses replication of the vector in the absence of HIV.

76. A method for eradication of HIV from an HIV-infected subject according to claim 75, wherein the vector is a lentiviral vector.

77. A method for eradication of HIV from an HIV-infected subject according to claim 76, wherein the lentiviral vector is a feline immunodeficiency virus (FlV)-based vector.

78. A method for eradication of HIV from an HIV-infected subject according to claim 77, wherein the FIV-based vector comprises a modified wild type FIV strand and an FIV cargo strand.

79. A method for eradication of HIV from an HIV-infected subject according to claim 78, wherein the FIV cargo strand comprises the nucleic acid sequence encoding the dual-function114protein which inhibits infectious HIV from budding out of HIV-infected cells and represses replication of the vector in the absence of HIV.

80. A method for eradication of HIV from an HIV-infected subject according to claim 79, wherein the nucleic acid sequence on the FIV cargo strand that encodes the dual function protein is 2STOP.

81. A method for eradication of HIV from an HIV-infected subject, according to claim 75, wherein the dual-function protein is 2STOP.

82. A method for eradication of HIV from an HIV-infected subject according to claim 75, further comprising establishing which strain of HIV is to be treated.

83. A method for eradication of HIV from an HIV-infected subject according to claim 82, wherein establishing which strain of HIV is to be treated is determined prior to the delivering the vector.

84. A method for eradication of HIV from an HIV-infected subject according to claim 83, wherein establishing the strain of HIV comprises performing an analysis selected from the group consisting of ELIS As, Western Blot analysis, RT-PCR, and combinations thereof.

85. A method for eradication of HIV from an HIV-infected subject, comprising administering to the subject an effective dose of a vector comprised of a nucleic acid sequence encoding a dual function protein that represses vector replication by auto-inhibition in the absence of HIV and inhibits infectious HIV from budding from membranes of HIV-infected cells in the event of HIV infection.

86. A method for eradication of HIV from an HIV-infected subject according to claim 76, further comprising determining the effective dose by performing a T-cell count on the subject to establish the required number of transducing units, and wherein the effective dose of the lentiviral vector for in vivo gene delivery is 1 to 50 MOI per T-cell.

87. A method for eradication of HIV from an HIV-infected subject according to claim 86, wherein the vector is administered intravenously, intraarterially, intralesionally, percutaneously, subcutaneously, intramuscular, intrathecally, intraorbitally, intradermally, intraperitoneally, transtracheally, subcuticularly, by intrasternal injection, by inhalation or intranasal spraying, by endrotracheal route, or combinations thereof.

88. A method for eradication of HIV from an HIV-infected subject according to claim 78, wherein the vector further comprises adjunctive protective transgenes and auxiliary transgenes selected for the specific strain of infectious HIV to be treated.

89. The method of claim 88, wherein the adjunctive protective transgenes comprise CRISPR-based genome editing targeting one or more HIV genomic sequences for insertional disruption wherein the HIV genomic sequences are selected from a group including but not limited to gag, pol, env, tat, rev, nef, vif, vpr, vpu, vpx, or functional equivalents thereof.

90. A method for eradication of HIV from an HIV-infected subject according to claim 88, wherein the adjunctive protective transgenes comprise CRISPR-based gene editing technology for site-specific integration into a genomic safe harbor locus such as can be selected from the group consisting of AAVS1, CCR5, ROSA26, Hippl 1, and HPRT1, and functional equivalents thereof.

91. A method for eradication of HIV from an HIV-infected subject according to claim 88, wherein the adjunctive protective transgenes comprise shRNA sequences targeting HIV- encoded sequences selected from gag-pol, integrase, env, vif, vpu, vpr, rev, vpx, or combinations thereof.

92. A method for eradication of HIV from an HIV-infected subject according to claim 88, wherein the adjunctive protective transgenes comprise CRISPR-based gene editing system configured to direct integration of the vector cargo into the CCR5 locus of a human T-cell, thereby disrupting CCR5 expression and conferring resistance to CCR5-tropic HIV infection.

93. A method for eradication of HIV from an HIV-infected subject according to claim 88, wherein the CRISPR-based gene editing technology comprises one or more sequences that direct excision of HIV sequences from the genome of infected host cells.

94. The method of claim 75 wherein other viruses that might be targeted using adjunctive protective transgenes delivered by means of this lentiviral vector include but not are not limited to retroviruses, hepadnaviruses, flaviviruses, herpesviruses, filoviruses, arenaviruses, poxviruses, orthomyxoviruses, coronaviruses, paramyxoviruses, and parvoviruses, as well as related or emerging viral pathogens.

95. The method of claim 75, comprising the identification and characterization of a pathological condition in a patient, the method including performing one or more molecular assays or immunologic assays configured to detect disease-specific molecular targets and to determine sequence variations or biomarker profiles relevant to therapeutic targeting, wherein the pathological condition is selected from the group consisting of, but not limited to, monogenic genetic disorders, polygenic disorders with defined molecular drivers, hemoglobinopathies, lysosomal storage disorders, muscular dystrophies, cystic fibrosis, alpha-1 antitrypsin deficiency, familial hypercholesterolemia, metabolic enzyme deficiencies,neurodegenerative disorders, and cancers with identifiable genetic mutations amenable to gene-based therapeutic intervention.

96. A kit of parts capable of being assembled, comprising a vector for eradicating human immunodeficiency virus (HIV) from a human comprising a nucleic acid sequence encoding a dual function protein that represses replication of the vector in the absence of HIV and inhibits infectious HIV from budding from membranes of HIV-infected cells.

97. A kit according to claim 96, further comprising components for assessing the strain of HIV to be treated which comprise PCR primers, anti-HIV antibodies to identify HIV serotypes or combinations thereof.

98. A kit according to claim 97, further comprising reagents for assembling a patient-specific genetic strategy for preventing, treating, or ameliorating HIV infection in a subject.

99. A kit according to claim 96, wherein the kit is configured to enable CRISPR-Cas- mediated integration of a therapeutic transgene into a genomic safe harbor locus selected from the group consisting of the adeno-associated virus integration site 1 (AAVS1, PPP1R12C locus), the CCR5 locus, the ROSA26 locus, the Hippl 1 locus, the HPRT1 locus, and functional equivalents thereof.

100. A kit for delivering an immunologic agent for targeting HIV virions in a subject, according to claim 96, further comprising a vector encoding at least one immunologic agent selected from the group consisting of protein aptamers, nucleic acid aptamers, monoclonal antibodies (mAbs), monovalent antibodies (mAbs), antigen-binding fragments (Fabs), single-chain variable fragments (scFvs), camelid nanobodies, anti -idiotypic antibodies, bi- specific antibodies, engineered immuno-adhesins, combinations and functional equivalents thereof.

101. A kit according to claim 96, further comprising instructions for using the kit.

102. The kit according to claim 101, wherein the instructions provide for characterizing the lenti vector.

103. The kit according to claim 102, wherein characterizing the lentivector comprises procedures for identifying and characterizing a virus infecting the patient.

104. The kit according to claim 101, wherein the instructions further comprise procedures for identifying and characterizing a pathogen or pathological condition other than HIV.

105. A dual -function protein comprising a transcriptional repressor domain that represses vector replication, and a dominant negative domain that inhibits budding of HIV from membranes of HIV infected cells.117106. A dual-function protein according to claim 105, wherein the dual-function protein is 2STOPA and wherein the sequence encoding the transcriptional repressor domain of 2STOPA is 5’ of the sequence encoding the dominant negative domain.

107. A dual-function protein according to claim 106 wherein the nucleic acid sequence encoding 2STOPA is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, and functional equivalents thereof.

108. A dual-function protein according to claim 105 wherein the dual-function protein is 2STOPB and wherein the sequence encoding the dominant negative domain of 2STOPB is 5’ of the sequence encoding the transcriptional repressor domain.

109. A dual-function protein according to claim 108 wherein the nucleic acid sequence encoding 2STOPB is selected from the group consisting of SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, and functional equivalents thereof.118

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