HIV Excision Utilizing CRISPR/Cas9 Technology: Attacking the Proviral Quasispecies in Reservoirs to Achieve a Cure

Abstract

Recently several gene-editing technologies developed are being explored for their potential utility in providing new and unique treatments for HIV. One of these technologies is the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 system. This system is being explored for its utility against host genes important to HIV infection, namely the HIV coreceptor CCR5, and for excision of the integrated genome from infected cells by targeting selected genes or genomic regions, especially the HIV-1 promoter or long terminal repeat (LTR). One of the major hurdles with the development of this technology for use in patients is defining the LTR sequence spectrum within the viral quasispecies present in the integrated virus and how that effects the number of guide RNAs (gRNAs) required to completely excise all proviral genomes. In this study, the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort was utilized to demonstrate that [1] the predominant sequence of the integrated proviral LTR within the PBMC compartment shows a decrease in the amount of variation per year regardless of the type of therapy; [2] predominant HIV-1 LTR sequence undergoes continued genetic change with respect to the predominant genotype in these cells for at least 6 years while on effective suppressive ART; [3] using next generation sequencing (NGS), to demonstrate that 4 of the 8 patient samples examined could have a complete gRNA regimen designed to target all known quasispecies; and [4] length of HAART therapy may reduce the number of gRNA required to eradicate provirus as shown by NGS and gRNA design for longitudinal samples of patient A0017 in the CARES cohort. Overall, these studies demonstrate the feasibility of addressing at least one of the major technological challenges of CRISPR/Cas9-mediated HIV-1 proviral genome eradication involving the effective targeting of all viral quasispecies in a given patient sample.

Keywords: CRISPR/cas9; HIV; excision; guide RNA; memory T cells.

Figure 1. HIV LTR genetic variation and gRNA design in well-controlled patients

Consecutive visits were compared by individually aligning all sequences from each patient using the MUSCLE alignment tool [33] and calculating the number of variations between consecutive visits. The number of nucleotide changes per 100bp was plotted against the time since the baseline visit to determine the rate of accumulated variations. A: Line segments from each patient were generated from the longitudinal variation based on the anti-retroviral therapy (ART) status between consecutive visits using the following coding scheme: green line segments indicate a longitudinal visit in which the patient was naive to ART (21 patients), red line segments indicate off/non-adherent ART visits (39 patients), grey line segments indicate on/adherent ART visits (168 patients), and black line segments on/adherent ART (54 patients) with viral loads always below 100 copies per ml. The top panel depicts all longitudinal samples per patient. The bottom panel shows median and standard deviation for each group at each year. B: LTRs from 45 patients on/adherent ART and 31 patients with discontinuous ART for at least three consecutive visits were analyzed as in A. The trajectory of each patient is shown in grey with the median in red and the upper and lower quartiles in blue. C: Utilizing Roche 454 next generation sequencing (NGS), NGS on genomic DNA isolated from PBMCs of 6 patients and 8 samples was performed on a 4.4kb fragment of the HIV genome, as previously described [32]. The number of gRNAs and target position for each patient sequenced was determined by aligning the short-read sequences to the HXB2 genome using the BWA aligner [34] and a local implementation of the algorithm used by the CRISPR design tool [24]. 23-mer sliding windows were constructed by extracting all completely overlapping reads and checked for a PAM sequence; all windows with less than 50 overlapping reads were excluded. The minimal number of gRNAs required to cleave each targetable window was calculated by testing all possible gRNAs. D: NGS reads from patient 17 visit 3 were mapped to HXB2 using the BWA aligner [34] as described above and examined for percent conservation (green line) and number of gRNAs necessary for excision of all known quasispecies (red line) at every position of the LTR. A table of the position and number is provided below the graph.