Background
Insights gained from successfully treated individuals demonstrate that a definite HIV cure must purge the replication-competent HIV reservoir, whilst making CD4+ cells resistant to HIV reinfection. Due to its versatility, the CRISPR-Cas9 genome editing tool holds great potential to eliminate latent proviral DNA and mutate the viral co-receptor CCR5. Still, its safety and effectiveness are hindered by the lack of precise delivery to infected cells. Here, we present CD4-targeted lipid nanoparticles (LNPs) for CRISPR-Cas9 delivery to achieve viral excision and CCR5 knockout with greater efficacy and precision.
Methods
As primary human CD4 T cells do not readily take up cargo, we first optimized T cell mRNA transfection efficiency. First, we screened a LNP library to identify a lipid formulation with optimal properties including particle size, surface charge and mRNA encapsulation efficacy. Selected LNPs were then administered to various primary and immortalized T cells to select LNPs that efficiently bind and transfect T cells. In order to target this optimal LNP to HIV susceptible cells, we conjugated it to anti-CD4 nanobodies using biorthogonal click chemistry. Finally, we packaged sgRNA and Cas9 mRNA into our optimal LNP formulation and delivered it to T cells. We designed sgRNAs for CCR5 and LTR, to genetically edit the viral co-receptor in SupT1 cells, and to achieve viral excision in J-Lat 10.6 cells, respectively.
Results
First, we identified a LNP formulation with optimal physiochemical properties that enabled up to 100% binding to and transfection of SupT1, Jurkat and J-Lat 10.6 cells. Moreover, we showed up to 95% transfection of activated, and up to 25% of non-activated primary human CD4 T cells. Interestingly, conjugating the optimal LNP to anti-CD4 nanobodies enhanced the binding to primary human CD4 cells with ten-fold, compared to LNP alone. Next, our results showed successful gene editing in T cell lines. These CRISPR LNPs were able to mutate CCR5 in SupT1 cells, with an indel (insertion and deletion) frequency of 46%. Lastly, delivery of LTR CRISPR LNPs to J-Lat 10.6 cells resulted in up to 100% proviral excision and up to 80% indels in the HIV LTR.
Conclusions
Our results, demonstrating successful gene editing of the CCR5 co-receptor along with full-length provirus excision through targeted LNP delivery, provide proof of concept for CD4-targeted CRISPR LNPs as a genetic cure for HIV.