Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector for therapeutic and vaccine applications, but its low transduction efficacy currently limits its utility. The primary reasons for low transduction efficacy are as follows: (i) inefficient trafficking of the rAAV genome to the nucleus, (ii) inefficient host cell-mediated synthesis of double-stranded DNA from the single-stranded genome, and (iii) Toll like receptor (TLR) 9 responses. The goal of this proposal is to identify small molecules that increase the transduction efficacy of rAAV transduction by modulating any one of the barriers. One immediate application will be the use of these compounds in the rAAV-based gene-therapy against HIV developed in Dr. Michael Farzan's laboratory at The Scripps Research Institute. The rAAV vector expresses an antibody-like immunoadhesin (eCD4-Ig), a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, which binds to the HIV-1/simian immunodeficiency virus (SIV) envelope glycoprotein and protects macaques from infection. Increased transduction efficacy will decrease both the cost of treatment and the adaptive immune response to the viral capsid and expressed transgene that limits the use of rAAV vectors in humans. However, the significance of our studies goes beyond HIV-1 prophylaxis. These small molecules will be useful for other rAAV-based vaccines and therapeutics, such as vaccines developed for influenza, malaria, and dengue infection, or for the long-term delivery of biologics. In Phase I, we will (i) develop a high-throughput screening (HTS) assay to identify compounds that enhance rAAV transduction, (ii) identify transduction enhancers, (iii) broadly evaluate the mode of action, and prioritize hits for a Phase II study. Promising scaffolds will be subjected to a rational drug design program in Phase II, and will be advanced to IND enabling studies.
Public Health Relevance Statement: Public Health Statement: Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector that can be engineered to efficiently deliver and express a transgene. The current proposal is focused on discovering small molecules that will increase the transduction efficacy of rAAV vectors. The increased transduction efficacy of the rAAV-based gene-therapy vector will reduce cost of rAAV based vaccines and therapeutics and limit immune responses to the viral capsid or transgene product.
Project Terms: glycerophosphatase; alkaline phosphomonoesterase; Alkaline Phosphatase; Antibodies; Biologic Assays; Bioassay; Assay; Biological Assay; cDNA; Complementary DNA; Capsid; cell culture; Cell Culture Techniques; cultured cell line; Strains Cell Lines; CellLine; Cell Line; Nucleus; Cell Nucleus; Cell Body; Cells; Drug Design; Embryonic; Embryo; Engineering; Fluorescence; genetic therapy; gene-based therapy; Genetic Intervention; Gene-Tx; Gene Transfer Procedure; Gene Transfer Clinical; Gene Therapy Molecular Biology; DNA Therapy; gene therapy; Genome; Glycoproteins; Goals; Grant; Virus-HIV; Lymphadenopathy-Associated Virus; LAV-HTLV-III; Human T-Lymphotropic Virus Type III; Human T-Cell Lymphotropic Virus Type III; Human T-Cell Leukemia Virus Type III; Human Immunodeficiency Viruses; HTLV-III; Acquired Immunodeficiency Syndrome Virus; Acquired Immune Deficiency Syndrome Virus; AIDS Virus; HIV; human T lymphotropic virus III; human T cell leukemia virus III; Human immunodeficiency virus 1; Human Immunodeficiency Virus Type 1; HIV1; HIV-I; HIV-1; Modern Man; Human; In Vitro; Infection; Influenza; influenza infection; flu infection; Grippe; Laboratories; Lead; heavy metal lead; heavy metal Pb; Pb element; Libraries; Ligands; Macaca; Macaque; Production; Proteins; Public Health; Research Institute; Research Proposals; Role; social role; Safety; Signal Transduction; biological signal transduction; Signaling; Signal Transduction Systems; particle; Animal Model; model organism; model of animal; Animal Models and Related Studies; trafficking; response; high throughput screening; High Throughput Assay; mimetics; TLR9 gene; toll-like receptor 9; TLR9 receptor; TLR9 protein; Binding; Molecular Interaction; protein expression; Alpha Cell; a-cell; Glucagon Secreting Cell; Glucagon Cell; small molecule; CCR5 gene; HIV-1 Fusion Co-Receptor Gene; HIV-1 Fusion Co-Receptor; Chemokine (C-C) Receptor 5 Gene; Chemokine (C-C) Receptor 5; Chemokine (C-C Motif) Receptor 5; CMKBR5 Gene; CMKBR5; CKR5 Receptors; CKR5 Gene; CKR5; CKR-5 Gene; CKR-5; CHEMR13 Gene; CHEMR13; CD195 Antigen Gene; CD195 Antigen; CCR5 Receptors; CCR5 Protein; CCR5; CCR-5 Gene; CCR-5; CCCKR5 Gene; CCCKR5; CC-CKR5; CC-CKR-5 Gene; CC-CKR-5; CC Chemokine Receptor 5; C-C Chemokine Receptor Type 5 Gene; C-C Chemokine Receptor Type 5; C-C CKR-5 Gene; C-C CKR-5; Dose; cytotoxic; Detection; Gene Transduction Vectors; Gene Therapy Vectors; Gene Transduction Agent; Gene Delivery; developmental; Development; vector; cost; designing; design; Recombinant adeno-associated virus (rAAV); rAAV; Recombinant adeno-associated virus; transgene expression; phase 2 study; phase II study; screening; Dengue Infection; dengue viral infection; Dengue virus infection; DENV infection; malaria infection; malaria-infected; adaptive immune response; Intracellular Communication and Signaling; Cell Signaling; Cell Communication and Signaling; SIV; Simian Immunodeficiency Viruses; Time; VAC-TX; Vaccine Therapy; Vaccines; virus genome; Viral Genome; General Viruses; Virus; Measures; Enhancers; Mediating; Treatment Cost; base; virus envelope; Prophylaxis; Prophylactic treatment; Phase; Reporter Genes; Proteosome; Proteasome Endopeptidase Complex; Proteasome; Prosome; Multicatalytic Proteinase; Macroxyproteinase; Macropain; 20S Proteosome; 20S Proteasome; 20S Core Proteasome; 20S Catalytic Proteasome; multicatalytic endopeptidase complex; Measurement; T8 Lymphocytes; T8 Cells; CD8-Positive Lymphocytes; CD8+ T-Lymphocyte; CD8+ T cell; CD8 lymphocyte; CD8 T cells; CD8 Cell; CD8-Positive T-Lymphocytes; Funding; Transgenes; Immune response; immunoresponse; host response; Immunological response; Therapeutic; ds-DNA; dsDNA; Double-Stranded DNA; Shapes; Reporter; scaffold; scaffolding; programs; Intramuscular; Route; neutralizing antibody; Viral; Nuclear
