Thursday, November 21, 2024
11/21/2024
PROJECT SUMMARY: In aging HIV-infected populations comorbid diseases are important determinants of morbidity and mortality. HIV patients die of non-AIDS comorbidities almost a decade earlier that their non-HIV counterparts. cART is unable to eradicate HIV due to established HIV reservoirs. Mounting evidence suggests that exhaustion of the latent viral reservoirs leads to either a complete cure (sterilizing cure) or a remission (functional cure). HIV establishes reservoirs at several anatomical sites like peripheral blood, CNS, lung, etc. Gene therapy has shown enormous promise but its application towards HIV cure is limited due to limitations like viral escape, off-target effects and most importantly delivery and bioavailability to infected cells. Gene editing using CRISPR/Cas9 has tremendous potential in HIV gene therapy and reports have shown excision of HIV from infected cells. However, CRISPR based HIV excision is vulnerable to viral escape as well as off-target effects. P-TEFb, a heterodimer of cyclinT1 and CDK9 is recruited by HIV Tat and plays a pivotal role in releasing the elongation block to allow transcription to proceed from the HIV LTR. Interaction of Tat with CyclinT1 is critical to recruit PTEF-b to turn ON HIV transcription, while CyclinT1 can be substituted by other cyclins for cellular transcription. This criticality for HIV transcription and redundancy for cellular transcription makes CyclinT1 an ideal cellular factor to block HIV with minimal effects on cellular transcription. Inhibition of Cyclin T1 has been shown to completely inhibit HIV transcription. Hence a CRISPR mediated inactivation of CyclinT1 will completely block HIV transcription, locking it in an inactive form in the cell thereby effecting a functional cure. Effecting CRISPR-mediated CyclinT1 knockdown only in HIV infected cells provides an additional insulation from any toxicity or off-target effects. We will restrict CyclinT1 knockdown only in infected cells by co-expressing gRNA targeting Cyclin T1 along with Cas9 from a Pol II mono-promoter based single transcription cassette using our patented LTR-hsp fusion promoter co-expression cassette. Expression is self- limiting as the co-expression cassette is also dependent on HIV Tat-CyclinT1 interaction thereby preventing generation of adaptive immune responses. Upon CyclinT1 knockdown, expression of gRNA and Cas9 will cease from the fusion promoter. We will combine this specific expression with lentiviral vector based in vivo delivery using a regimen that has demonstrated delivery and transduction of bone marrow stem cells and PBMCs. In Aim 1, will determine the efficacy of our self-limiting HIV inducible gRNA-Cas9 co-expression cassette to block and lock HIV proviral transcription in primary cell models of infection and HIV latency. Aim2 we will test the pharmacokinetics, toxicity and efficacy of lentiviral vector based in vivo delivery of our HIV inducible CRISPR targeting CyclinT1 in small animal models of HIV infection and latency. This application responds to key objectives of RFA-AI-20-076 by proposing “in vivo gene therapeutic strategies with selective regulation of genetic payload expression to maximize on-target efficacy and minimize off-target effects.”
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