Background
A major challenge in curing HIV is eliminating latent integrated proviral DNA from CD4+ T and myeloid cells. Recent clinical studies (EBT-101) using adeno-associated viral (AAV) vectors to deliver CRISPR for HIV eradication remained unsuccessful likely due to the limitations of AAV vectors, highlighting the need for alternative delivery systems like lipid nanoparticles (LNPs). To this end, we created a lymphoid tissue-targeted LNP that includes a cyclic pentapeptide to selectively bind to CXCR4, a co-receptor that HIV uses to infect CD4+ T cells, aiming to eliminate HIV from latently infected tissue/cells reservoir more effectively.
Methods
CXCR4-targeting peptide decorated LNP (T-LNP) was formulated by using microfluidic mixing. The receptor specificity was shown by CXCR4 inhibition assay. IVIS was performed to demonstrate lymphoid tissue targeting on humanized mice (hu-mice). The intact proviral DNA assay (IPDA) assay was utilized to test CRISPR gRNA delivery and viral elimination efficiency in latently HIV-infected lymphocytic cell lines (JLat 8.4, Jurkat E6), human peripheral blood lymphocytes (PBLs), and lymphoid tissue from HIV-infected, ART-treated hu-mice.
Results
T-LNP and a control LNP without CXCR4 targeting (C-LNP) were tested in primary CD4+ T cells, cell lines, and hu-mice. Unlike C-LNP, T-LNP in humanized mice showed a significant shift in tissue targeting from the liver to the spleen, a primary HIV reservoir. T-LNP showed significantly higher viral excision efficiency in latently infected cells whose specificity was confirmed by a receptor inhibition assay through increasing concentrations of a CXCR4 inhibitor. T-LNP achieved nearly 80% viral DNA excision in HIV-1-infected PBLs, compared to ~7% with C-LNP. In HIV-1-infected, ART-treated humanized mice, T-LNP showed ~60% CRISPR efficacy in splenic tissue and blood, while C-LNP showed ˂5% efficacy, analyzed by the IPDA.
Conclusions
T-LNP demonstrated lymphoid cell-targeted CRISPR/Cas9 delivery. This delivery system was effective in eliminating HIV-1 from infected CD4+ T cells. Furthermore, T-LNPs have shown superior excision efficiency compared to C-LNP in eliminating HIV-1 DNA from hu-mice infectious reservoirs. These data suggest that T-LNP has the potential to achieve a functional HIV-1 cure.