Background:
A principal limitation in achieving HIV cure rests, first, in locating and then eliminating integrated proviral DNA from viral target cells (CD4+ T and myeloid cells). In recent reports, the development of viral vector to deliver clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs (gRNAs) provided a proof of concept that HIV-1 elimination could be achieved. Our goal is to extend and improve those results by creating: (1) multiple viral exons (gp41, tat, and rev) gRNAs; (2) viral tissue reservoir targeted lipid nanoparticles (LNPs); and (3) decorating LNPs with CXC chemokine receptor-4 (CXCR4) for targeting cellular reservoir. The biodistribution of LNPs was evaluated in humanized mice. We posit that effective gRNA delivery and, subsequently, viral DNA elimination can be achieved with our approach.
Methods:
A library of gRNAs was designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41). These were derived from a consensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. CXCR4-targeting cyclic-peptide (CycPep) was conjugated with PEG-lipid to make DSPE-PEG-CycPep and LNP was formulated by microfluidic mixing. The specificity of the CXCR4 receptor was shown by blocking the CXCR4 receptor to demonstrate lymphoid targeting. CRISPR gRNA delivery and excision were tested in lymphocytic (JLat 8.4, Jurkat E6) and monocytic (U1) cell lines and confirmed in primary human lymphoblasts.
Results:
A compositionally unique CXCR4 targeted LNP (T-LNP) was formulated with CRISPR gRNA-Cas9, D-Lin-MC3-DMA, DOPE, DOPS, DSPE-PEG-CycPep, DMG-PEG and β-sitosterol. T-LNP showed a significant shift in tropism in hu-mice from the liver to the spleen – one of the major HIV reservoirs. Further analysis revealed a substantially high mRNA expression in primary CD4+ T cells compared to nontargeted LNP. T-LNP demonstrated nearly 60% excision of viral DNA in lymphocytic cell lines; however, it was only 15% in myeloid cells. Moreover, in the presence of the CXCR4 blocker, T-LNP demonstrated decreased DNA excision, confirming CXCR4-specificity as compared to the control- LNP which lacked CXCR4 targeting.
Conclusions:
This work introduces a novel, non-viral delivery system to target HIV reservoirs. Our preliminary work shows that CXCR4-targeted LNP reached HIV reservoirs and enabled excision of integrated latent HIV-1 DNA, potentially leading to its elimination.
