RNA interference (RNAi) is a recently discovered gene-silencing phenomenon mediated by double stranded short interfering RNA (siRNA) that guide mRNA degradation in a sequence specific fashion. We have shown that siRNAs targeting cellular and viral genes involved in HIV life cycle can dramatically suppress virus replication in human cell lines and primary cells. Since RNAi is critically dependent on nucleotide sequence match, the propensity of HIV for sequence mutations is an impediment for using the technology as therapy against the virus. To overcome this limitation, we have identified optimal siRNA targets in highly conserved regions of the HIV genome and showed that these shRNAs can protect primary CD4 T cells from HIV-1 primary isolates within clade B or across multiple clades. In vivo delivery of siRNA/shRNA is the other major hurdle for translation of this promising new technology into the clinic. To use siRNA as a drug it will be critical to develop a method to introduce siRNA into primary T cells and macrophages. This is a major challenge because T cells take up siRNA very poorly even by in vitro transfection. We have recently shown that targeted delivery of siRNA to this difficult cell type can be accomplished by using a single chain antibody fused to protamine, which is a highly basic polypeptide that can bind siRNA. Further, we have also used a novel immunoliposome conjugated to 2 different antibodies that recognize LFA-1 either in the closed or open activated conformation to differentially deliver siRNA to all T cells irrespective of the activation status or specifically to activated T cells only. One other problem in a chronic infection like HIV is the need for long-term therapy. To this end, we have developed lentiviral vectors to endogenously express shRNAs and showed its potential to derive HIV-resistant progeny by transduction of CD34+ hematopoietic stem cells (HSCs). We now propose to optimize delivery methods for potential RNAi-based interventions in HIV infection in a humanized mouse model, using the novel SCID/NODIL2r?c-/- mouse strain developed by our collaborator Dr. Leonard Shultz at the Jackson Laboratory. The common gamma chain null SCID/Hu mice support complete development of human immune system after engraftment with purified CD34+ hematopoietic stem cells and reconstituted animals infected with HIV-1 show sustained and high levels of viral replication. Thus, the model provides us with a unique experimental system in which to optimize our delivery strategies. Our specific objectives are first to further refine the antibody/protamine fusion protein- and immunoliposome- mediated naked siRNA delivery to resting and/or activated T cells using antibodies to CD7 (a pan T cell molecule) or LFA-1 in open or closed conformation. Our next goal will be to test the in vivo efficacy of both delivery strategies in NOD/Lt-scid IL2r?null mice humanized either by PBL transplantation (SCID/hu-PBL) or by engrafting with cord blood CD34+ hematopoietic stem cells. Finally, we will reconstitute NOD/Lt-scid IL2r?null mice with lentivirus-transduced CD34+ human HSCs and test establishment of long-term HIV resistance. ? ? ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI071882-01A2
Application #
7339361
Study Section
AIDS Discovery and Development of Therapeutics Study Section (ADDT)
Program Officer
Gupta, Kailash C
Project Start
2007-06-15
Project End
2008-05-31
Budget Start
2007-06-15
Budget End
2008-05-31
Support Year
1
Fiscal Year
2007
Total Cost
$408,701
Indirect Cost
Name
Immune Disease Institute, Inc.
Department
Type
DUNS #
059709394
City
Boston
State
MA
Country
United States
Zip Code
02115
Choi, Jang-Gi; Bharaj, Preeti; Abraham, Sojan et al. (2015) Multiplexing seven miRNA-Based shRNAs to suppress HIV replication. Mol Ther 23:310-20
Abraham, Sojan; Pahwa, Rajendra; Ye, Chunting et al. (2012) Long-term engraftment of human natural T regulatory cells in NOD/SCID IL2r?c(null) mice by expression of human IL-2. PLoS One 7:e51832
Joshi, Anjali; Garg, Himanshu; Ablan, Sherimay et al. (2011) Targeting the HIV entry, assembly and release pathways for anti-HIV gene therapy. Virology 415:95-106
Kim, Sang-Soo; Peer, Dan; Kumar, Priti et al. (2010) RNAi-mediated CCR5 silencing by LFA-1-targeted nanoparticles prevents HIV infection in BLT mice. Mol Ther 18:370-6
Subramanya, Sandesh; Kim, Sang-Soo; Abraham, Sojan et al. (2010) Targeted delivery of small interfering RNA to human dendritic cells to suppress dengue virus infection and associated proinflammatory cytokine production. J Virol 84:2490-501
Subramanya, Sandesh; Kim, Sang-Soo; Manjunath, N et al. (2010) RNA interference-based therapeutics for human immunodeficiency virus HIV-1 treatment: synthetic siRNA or vector-based shRNA? Expert Opin Biol Ther 10:201-13
Subramanya, Sandesh; Armant, Myriam; Salkowitz, Janelle R et al. (2010) Enhanced induction of HIV-specific cytotoxic T lymphocytes by dendritic cell-targeted delivery of SOCS-1 siRNA. Mol Ther 18:2028-37
Manjunath, N; Wu, Haoquan; Subramanya, Sandesh et al. (2009) Lentiviral delivery of short hairpin RNAs. Adv Drug Deliv Rev 61:732-45
Kumar, Priti; Ban, Hong-Seok; Kim, Sang-Soo et al. (2008) T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice. Cell 134:577-86