HIV-1 mRNA Knockdown with CRISPR/Cas9 Enhances Neurocognitive Function

Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 mRNA from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 μL) of CRISPR/Cas9 for 72 hours. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n=5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the mPFC. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1 associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 partially restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.


INTRODUCTION
The current treatment regimen for HIV-1 seropositive individuals includes a cocktail of two to four drugs, collectively known as combination antiretroviral therapy (cART), which act to suppress viral replication (e.g., Gulick et al., 1997).cART revolutionized the treatment of HIV-1 and transformed the disease into a chronic, manageable condition, whereby HIV-1 seropositive individuals have life expectancies similar to those of seronegative persons (Marcus et al., 2020).Nevertheless, disease management is challenged by poor adherence to cART, which is associated with viral rebound (e.g., Maina et al., 2020).Furthermore, despite treatment with cART, HIV-1 seropositive individuals are disproportionately a icted by comorbidities (Marcus et al., 2020; e.g., Liver Disorders: Morales et al., 2022; Cardiovascular Disease: Alonso et al., 2019, Touloumi et al., 2020) and exhibit high rates of neurocognitive impairments (e.g., Wang et al., 2020).In light of these observations, scientists have been on a quest to develop a functional cure (i.e., long-term HIV-1 control in the absence of cART) for HIV-1.
Elimination of HIV-1 viral reservoirs, which are established early in the course of infection (Gantner et al., 2023) and persist despite cART (e.g., Chun et al., 1997;Finzi et al., 1997;Wong et al., 1997), presents a key challenge to the development of a functional cure.A viral reservoir, as de ned by Eisele and Siliciano (2012), includes infected cell populations that allow long-term persistence (i.e., years) of replicationcompetent HIV-1 in HIV-1 seropositive individuals on cART.Although the primary HIV-1 viral reservoir resides in memory CD4 + T cells, there is compelling evidence for the existence of additional viral reservoir sites, including the central nervous system (CNS; for review, Churchill et al., 2016, Wallet et al., 2019).Microglia, in particular, are the resident immune cells of the CNS whose lifespan is approximately 4.2 years (Réu et al., 2017).Further, the migration of HIV-1-infected monocytes across the blood-brain barrier (for review, Williams et al., 2012) results in the infection of microglia evidenced by the presence of replication-competent HIV-1 (i.e., HIV-1 DNA) in both untreated (Thompson et al., 2011) and virally suppressed (Ko et al., 2019) HIV-1 seropositive individuals.The ability of microglia to harbor HIV-1 DNA, in conjunction with their long lifespan, meets the criteria for a viral reservoir based on the de nition established by Eisele and Siliciano (2012).Eradication of HIV-1 from the CNS, therefore, is likely to be of fundamental import for the successful implementation of a functional cure strategy.
Three broad strategies have been pursued to completely eradicate the HIV-1 genome, including stem cell transplantation (Hutter et al., 2009;Gupta et al., 2019;Jensen et al., 2023;Hsu et al., 2023), "shock and kill" approaches (e.g., for review, Ait-Ammar et al., 2020), and gene editing (e.g., Hu et al., 2014;Qu et al., 2013).Successful eradication of the HIV-1 genome has been achieved in four individuals, whereby three individuals received a CCR5Δ32 homozygous allogeneic adult stem cell transplant (Hutter et al., 2009;Gupta et al., 2019;Jensen et al., 2023) and one adult was cured via a CCR5Δ32/Δ32 haplo-cord transplant (Hsu et al., 2023).Despite the recent success of a combined haploidentical and cord blood transplant, which has the potential to increase donor cell availability, widespread implementation of this approach remains impractical.The "shock and kill" approach utilizes latency-reversing agents (LRA; e.g., Tat-R5M4: Geng et al., 2016) to reactivate HIV-1 transcription, thereby enabling the immune system to kill latently infected cells.Although treatment with LRAs induces reactivation of viral transcription in latently infected cells, the viral reservoir size is not reduced in a clinically meaningful manner (e.g., Rasmussen et al., 2014;Gruell et al., 2022).Further, the CNS presents an additional obstacle for the "shock and kill" approach, as it may induce unintended adverse effects, including harmful neuroin ammation (Gama et al., 2017).Programmable nuclease-based gene editing utilizing zinc nger (Qu et al., 2013) or clustered, regularly-interspaced, short palindromic repeats (CRISPR)/ CRISPR-associated 9 (Cas9) nucleases (e.g., Hu et al., 2014;Kaminski et al., 2016a;Dash et al., 2019), however, represent a promising strategy to eradicate the HIV-1 genome from infected cells.CRISPRs, which were rst identi ed in the genome of Escherichia coli (Ishino et al., 1987), are a family of repetitive deoxyribonucleic acid (DNA) sequences characterized by direct repeats interspaced by similarly sized non-repetitive elements clustered in at least one loci on the chromosome.Upstream of the CRISPR array are CRISPR-associated (cas) genes that encode proteins with nuclease and helicase domains (e.g., Cas9: Makarova et al., 2002), The Cas9 protein, in particular, contains an HNH and RuvC-like nuclease domain; distinct active sites that are utilized to cleave double-stranded DNA (dsDNA; Jinek et al., 2012;Gasiunas et al., 2012).Fundamentally, co-expression of Cas9 and custom-engineered guide ribonucleic acid (gRNA) molecule(s) induces highly e cient site-speci c alterations in the target DNA affording a novel tool for gene editing (Jinek et al., 2012;Cong et al., 2013;Jinek et al., 2013;Mali et al., 2013).Indeed, Cas9-mediated genome editing affords a novel strategy to excise the integrated HIV-1 proviral genome in vitro and in vivo (for review, Bhowmik & Chaubey, 2022).The development of single gRNA molecules to target the anking HIV-1 long terminal repeat (LTR) sequences provided a fundamental proof-of-concept, whereby treatment cleaved viral DNA in vitro and inhibited viral gene expression (Ebina et al., 2013;Hu et al., 2014;Kaminski et al., 2016a;Yin et al., 2016).The potential for InDel generation, and thus an "escape mutant", with a single gRNA (Wang G et al., 2016;Wang Z et al., 2016), however, necessitates a multiplex gRNA con guration; a con guration that also e caciously eradicates the integrated HIV-1 proviral genome in vitro (e.g., Hu et al., 2014;Kaminski et al., 2016a;Yin et al., 2016).Fundamentally, delivery of the Cas9/gRNA system using an adeno-associated virus (AAV) vector excises the HIV-1 provirus in vivo (Kaminski et al., 2016b;Yin et al., 2017;Dash et al., 2019).The e cacy of Cas9/gRNAs for the excision of constitutively expressed HIV-1 viral proteins from the CNS, however, remains understudied.Thus, complementary in vitro and in vivo aims were undertaken to address this fundamental knowledge gap using the HIV-1 transgenic (Tg) rat.The HIV-1 Tg rat, originally reported by Reid et al. (2001), expresses HIV-1 viral proteins constitutively throughout development (Peng et al., 2010;Abbondanzo & Chang, 2014), whereby HIV-1 mRNA is predominantly expressed in microglia in a restricted, regionspeci c manner (Li et al., 2021).First, the utility of the Cas9/gRNA system to excise HIV-1 viral proteins in vitro from primary mixed glia was established using a dose-response experimental paradigm.Second, the e cacy of the Cas9/gRNA system for HIV-1 mRNA excision in vivo was evaluated.Third, the clinical relevance of HIV-1 mRNA knockdown for mitigating neurocognitive impairments was examined using a longitudinal evaluation of temporal processing; temporal processing is a potential neurobehavioral mechanism underlying higher-order cognitive processes (McLaurin et al., 2019a).Establishing the susceptibility of HIV-1-infected mixed glia to gene editing via AAV9-CRISPR/Cas9 is fundamental to the widespread implementation and success of the therapeutic approach.

Experimental Design
A schematic of the experimental design for the complementary in vitro and in vivo aims is illustrated in Fig. 1.

Animals
For Experiment #1: In Vitro Excision of HIV-1 mRNA, breeding of Fischer F344/N HIV-1 Tg pups was conducted at the University of South Carolina, whereby a Fischer F344/N control female (Envigo Laboratories Inc., Indianapolis, IN, USA) was paired with an HIV-1 Tg male.Primary mixed glia were isolated from no more than one male and one female HIV-1 Tg rat from each litter to preclude violation of the assumption of independence.
For Experiment #2: In Vivo Excision of HIV-1 mRNA and Experiment #3: Clinical Import of CRISPR/Cas9 Excision In Vivo male and female HIV-1 Tg and control (Fischer F344/N) animals were procured from Envigo Laboratories during early adolescence.Animals were pair-housed with animals of the same sex for the duration of the study.
During both experiments, HIV-1 Tg and control rats had ad libitum access to rodent food (Pro-Lab Rat, Mouse, Hamster Chow #3000) and water.Animals were housed in AAALAC-accredited facilities with environmental conditions targeted at 21˚C ± 2˚C, 50% ± 10% relative humidity, and a 12-h light:12-h dark cycle with lights on at 700 h (EST).Guidelines established by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals were utilized for the maintenance of all animals.The University of South Carolina Institutional Animal Care and Use Committee approved the project protocol under Federal Assurance (#D16-00028).

Primary Mixed Glial Cell Cultures
The prefrontal cortex was dissected from male (n = 4) and female (n = 4) neonatal (i.e., Postnatal Day 1-3) HIV-1 Tg rat pups for the isolation of mixed glia.Following dissection, brain tissue was transferred into a 15 mL centrifuge tube with Hank's balanced salt solution (HBSS) buffered with 10 mM HEPES (GIBCO Life Technologies, Grand Island, NY, USA) and 0.25% Trypsin/EDTA (GIBCO Life Technologies); the centrifuge tube was incubated at 37˚C in 5% CO 2 /95% room air-humidi ed incubator for 20 minutes.Tissue was dissociated using trituration and transferred into a 75 mm poly-L-lysine coated ask with DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS).DMEM/F12 medium supplemented with 10% FBS was changed the day after isolation and supplemented every three to seven days.Upon reaching at least 90% con uency, mixed glia were reseeded into poly-L-lysine coated 8-well CultureSlides (Corning, Corning, NY, USA) for experimentation.Cultures were maintained in the incubator (i.e., 37˚C in 5% CO 2 and 95% humidity) at all times.

CRISPR/Cas9 Plasmid and Treatment
A detailed description of the guide RNA (gRNA) design, construction of the CRISPR/Cas9 expression plasmid, and adeno-associated virus 9 (AAV 9 ) vector have been previously published (Hu et al., 2014).In brief, gRNAs with high guide e ciency (i.e., to maximize on-target activities in the HIV-1 genome) and speci city (i.e., to minimize off-target effects in the human genome) were selected based on screening using the Broad Institute gRNA designer tool; screening which yielded two gRNAs targeting the HIV-1 LTR promotor region and the gag gene.A plasmid carrying the SaCas9 endo-nuclease gene under the CMV promoter and gRNAs was packaged into an AAV 9 serotype (Vigene Biosciences Inc., Milton Park, Abingdon, UK).An AAV 9 delivery vector was selected for the delivery of CRISPR/Cas9 given its robust transduction e ciency into the central nervous system (Foust et al., 2009).Cortical mixed glial cell cultures were treated for 72 hours with one of seven doses of AAV 9 -CRISPR/Cas9 (i.e., 0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL).
Upon continuation of the in situ hybridization assay, cells were rehydrated using a decreasing EtOH gradient (i.e., 70% EtOH for 2 minutes, 50% EtOH for 2 minutes) followed by PBS (10 minutes).A hydrophobic barrier was created around each well using the ImmEdge Hydrophobic Barrier PAP Pen (Vector Laboratories, Burlingame, CA, USA) and allowed to dry completely.After being rinsed with PBS, the RNAscope Protease III reagent (Advanced Cell Diagnostics, Inc.) was added to cells in each well and incubated in the HybEZ Humidity Control Tray (Advanced Cell Diagnostics, Inc.).Slides were washed twice by agitating them in PBS.

HIV-1 mRNA Quanti cation
Multiple z-stack images were obtained from high con uency areas, as evidenced by DAPI staining, in each well using a Nikon TE-200E confocal microscope system controlled by Version 3.81b of Nikon's EZ-C1 software.Images were captured at 60x using an oil objective with a numerical aperture of 1.4 and zplane intervals of 0.15 µm.Fluorophore excitation of the HIV-1 mRNA probe and DAPI staining were accomplished using an argon helium neon laser (Emission: 488 nm) and HeNe helium-neon (Emission: 633 nm), respectively.
HIV-1 mRNA exhibited a "discrete dots" (Li et al., 2018) staining pattern affording an opportunity to quantify the number of HIV-1 mRNA.Z-stack images were blinded to prevent experimental bias.The number of HIV-1 mRNA signals were counted from two z-stack images per well based on established selection criteria (i.e., high cell con uency, bright HIV-1 mRNA signal).To preclude violation of the assumption of independence, the number of HIV-1 mRNA signals from the two z-stack images per well was averaged (Denenberg, 1984;Wears, 2002).
Experiment #2: In Vivo Excision of HIV-1 mRNA Retro-orbital CRISPR/Cas9 Inoculation Adult male and female HIV-1 Tg animals were randomly assigned to receive retro-orbital injections of either CRISPR/Cas9 (i.e., AAV9-SaCas9-sgRNA-HIV-LTR1/GagD, 4.7 x 10 12 gc/ml; n = 4; Male: n = 2, Female: n = 4) or saline (n = 4; Male: n = 2, Female: n = 4).After inhalant anesthesia was induced with sevo urane, the animal was placed laterally with the injection-eye facing upwards.A 1cc tuberculin syringe with a 26G needle was slowly inserted into the medial canthus of the eye at a 45˚ angle to the nose.50 µL of CRISPR/Cas9 or saline was gently injected into the retro-orbital vessels.Animals were allowed to recover in a heat-regulated warm chamber.

Tissue Preparation
Approximately two weeks after CRISPR/Cas9 inoculation, anesthesia was induced using 5% sevo urane, and HIV-1 Tg rodents were humanely sacri ced.Within ve minutes of sacri ce, the brain was harvested from the skull and frozen in liquid nitrogen for 15-sec.The brain was sliced into coronal sections using a cryostat (30 µm) and transferred onto slides (SuperFrost Plus Slides, Thermo Fisher Scienti c, Hampton, NH, USA) for in situ hybridization.
In Situ Hybridization using RNAscope HIV-1 mRNA expression in the mPFC was evaluated using a highly speci c and sensitive RNA in situ hybridization technique.Minor modi cations to the RNA in situ hybridization technique, described in detail by Li

Apparatus
The startle platform (SR-Lab Startle Re ex System, San Diego Instruments, Inc., San Diego, CA) and Plexiglas test cylinder were enclosed within a double-walled isolation cabinet (External Dimensions: 81 x 81 x 116 cm; Industrial Acoustic Company, Inc., Bronx, NY) that provided 30 dB(A) of sound attenuation.In the absence of any stimuli, the ambient sound level within the isolation cabinet was 22 dB(A).Auditory prepulse and startle stimuli were presented using a high-frequency loudspeaker (Model #40-1278B, Radio Shack, Fort Worth, TX) that was mounted 30 cm above the Plexiglas test cylinder.A 22 lux white LED light, which was mounted on the wall of the isolation chamber, was utilized to present visual prepulse stimuli.A de ection of the Plexiglas test cylinder was produced by the animal's response to the auditory startle stimulus.Response signals were converted into analog signals by a piezoelectric accelerometer integral to the Plexiglas test cylinder, digitized (12-bit A to D), and saved to a hard disk.

Prepulse Inhibition of the Auditory Startle Response
Prior to an assessment of temporal processing, animals were habituated using the test procedure previously described (McLaurin et al., 2017a).Temporal processing was subsequently evaluated using a 30-minute cross-modal prepulse inhibition experimental paradigm (described in detail by McLaurin et al., 2017a).In brief, after a 5-minute acclimation period, the startling stimulus was presented for six trials; trials which were separated by a xed 10-sec intertrial interval.During the subsequent 72 testing trials, a visual or auditory prepulse was presented prior to the auditory startling stimulus at interstimulus intervals (ISIs) of 0, 30, 50, 100, 200, or 4000 msec; the 0 and 4000 msec ISI trials served as control trials to provide a reference auditory startle response (ASR) within the test session.Counterbalancing (i.e., ABBA) was implemented for the presentation of auditory and visual prepulse trials, while a Latin-square experimental design was utilized for the presentation of ISIs within 6-trial blocks.The intertrial interval for testing trials was variable, whereby it ranged from 15 to 25 sec.Mean peak ASR amplitude values were collected for subsequent analyses.

Statistical Analysis
Statistical analyses, including analysis of variance (ANOVA) and regression techniques, were conducted using SPSS Statistics 28 (IBM Corporation, Somer, NY) and GraphPad Prism 5 (La Jolla, CA), respectively.GraphPad Prism 5 was also used for the creation of gures.An α criterion of p ≤ 0.05 was set for the establishment of statistical signi cance.
Two approaches were utilized to evaluate the susceptibility of mixed glia to gene editing via CRISPR/Cas9 in vitro.First, statistical analyses were conducted on all primary mixed glia (n = 8).Second, when the overall analysis failed to reveal any statistically signi cant effects, complementary analyses that included a subset of animals were conducted.Indeed, the number of HIV-1 mRNA from primary mixed glia cultured from a subset of neonatal HIV-1 Tg rats was examined dependent upon which phenomena (i.e., excision at high (5.4 µL; n = 3) or low (1.8 µL; n = 2) dose of CRISPR/Cas9) they exhibited.Data were analyzed using a repeated measures ANOVA, whereby CRISPR/Cas9 dose served as a within-subjects factor.Two CRISPR/Cas9 doses were missing from primary mixed glia cultured from one neonatal HIV-1 Tg rat exhibiting signi cant excision when treated with 5.4 µL of CRISPR/Cas9; mean imputation was utilized for the repeated-measures ANOVA.Complementary linear regression analyses were also conducted.
The in vivo e cacy of CRISPR/Cas9 was evaluated using a univariate ANOVA, whereby the number of HIV-1 mRNA in the mPFC served as the dependent variable of interest.Treatment served as the betweensubjects factor.
Further, the clinical relevance of HIV-1 mRNA knockdown for mitigating neurocognitive impairments was evaluated using linear regression analyses, whereby two dependent variables of interest (i.e., Startle Response, PPI) were derived from the ISI function (McLaurin et al., 2019b) and examined using a priori planned comparisons.Speci cally, startle response was calculated as an average between the 0 and 4000 msec ISI trials, whereas the area of in ection of the ASR amplitude response curve was calculated as an index of PPI (McLaurin et al., 2016).Furthermore, the genotype de cit (i.e., Control vs. HIV-1 Tg Saline) and effect of CRISPR/Cas9 (i.e., Control vs. HIV-1 Tg CRISPR/Cas9) were established using a priori planned comparisons.Given that the viral vector HM4d had no statistically signi cant effect on either of the dependent variables of interest, the control data presented and analyzed were collapsed across viral infusion.
Second, HIV-1 mRNA was e caciously excised from mixed glia cultured from two neonatal HIV-1 Tg rat pups after treatment with low (i.e., 1.8 µL) doses of CRISPR/Cas9 (Fig. 2D).From 0 µL to 1.8 µL, the number of HIV-1 mRNA signals, expressed as a percentage change relative to the respective untreated control, decreased in a linear manner (Best-Fit Function: First-Order Polynomial, R 2 ≥ 0.81; Fig. 2E).The average excision e ciency of HIV-1 mRNA from primary mixed glia at the 1.8 µL dose of CRISPR/Cas9 Given incomplete excision, subsequent in vivo studies were conducted to establish the clinical relevance of HIV-1 mRNA knockdown.For example, the mitigation of HIV-1-associated neurocognitive disorders (HAND), which a ict approximately 50% of HIV-1 seropositive individuals (Wang et al., 2020), has the potential to improve the quality of life for HIV-1 seropositive individuals.Therefore, following retro-orbital inoculation with CRISPR/Cas9, the viral vector HM4d, or saline, temporal processing, a potential neurobehavioral mechanism underlying HAND (McLaurin et al., 2019a), was evaluated longitudinally using PPI.Two dependent variables of interest (i.e., Startle Response and Prepulse Inhibition) were examined using a priori planned comparisons to establish an HIV-1 genotype effect (i.e., Control vs. HIV-1 Tg Saline) and the magnitude of the treatment effect (i.e., Control vs. HIV-1 CRISPR/Cas9).Given that the viral vector HM4d had no statistically signi cant effect on either of the dependent variables of interest, the control data presented and analyzed were collapsed across viral infusion.
Constitutive expression of the HIV-1 transgene induces pronounced alterations in the development of temporal processing, indexed using startle response (Fig. 4A) and PPI (Fig. 4B).HIV-1 Tg animals treated with saline failed to exhibit any statistically signi cant development of either startle response or PPI from PD 60 to PD 120, whereby data were well-described by a horizontal line.In sharp contrast, the development of mean startle response and PPI in control animals was well-described by a rst-order polynomial (R 2 ≥ 0.84) and segmental linear regression (R 2 ≥ 0.96), respectively.Regression analyses, therefore, illustrate the prominent temporal processing de cit induced by HIV-1 viral protein exposure.
From PD 60 to PD 90, the developmental trajectory of startle response and PPI in HIV-1 Tg animals treated with CRISPR/Cas9 resembles that of control animals.Indeed, HIV-1 Tg animals inoculated with CRISPR/Cas9 exhibited a linear increase in startle response through PD 90 followed by a subsequent decrease (Best Fit Function: Segmental Linear Regression, R 2 ≥ 0.90).Overlapping 90% con dence intervals between HIV-1 Tg animals treated with CRISPR/Cas9 and control animals support statistically indistinguishable developmental trajectories.With regards to PPI, the linear increase from PD 60 to PD 90 observed in both HIV-1 Tg animals treated with CRISPR/Cas9 and control animals was well-described by a global t (i.e., First Order Polynomial: R 2 ≥ 0.78), whereby no statistically signi cant differences in the parameters of the function were observed (p ≥ 0.05).Taken together, even in the absence of complete excision, the knockdown of HIV-1 mRNA enhances neurocognitive function for a period of time.

DISCUSSION
Proof-of-concept studies support the susceptibility of mixed glia to gene editing via CRISPR/Cas9, whereby pronounced, albeit incomplete, excision of the HIV-1 viral genome was observed both in vitro and in vivo.In vitro, a subset (i.e., n = 5 out of 8) of primary mixed glia isolated from neonatal HIV-1 Tg rats exhibited dose-dependent decreases in the number of HIV-1 mRNA following CRISPR/Cas9 treatment.E cacious excision, de ned as at least a 35% decrease in the number of HIV-1 mRNA, from primary mixed glia occurred at either the 1.8 µL or 5.4 µL dose.In vivo, retro-orbital inoculation of CRISPR/Cas9 into the orbital venous plexus of HIV-1 Tg rats resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the mPFC.Nevertheless, excision of the complete HIV-1 viral genome may be unnecessary for the mitigation of HAND, as the developmental trajectory of temporal processing was partially restored in HIV-1 Tg animals treated with CRISPR/Cas9.Thus, independent of full viral eradication, gene editing via CRISPR/Cas9 may afford a novel therapeutic strategy for HAND.
Signi cant individual variability in the e cacy of CRISPR/Cas9 in primary mixed glial cultures (i.e., pronounced excision in only a subset (n = 5 out of 8 cultures) may be due, at least in part, to the dsDNA repair pathway utilized by cells.E cacious gene editing using CRISPR/Cas9 enzymes relies upon the cleavage of dsDNA at locations precisely de ned by custom-engineered gRNA molecule(s); genome integrity and the viability of the cell, however, are dependent upon proper repair of dsDNA breaks.dsDNA breaks are primarily repaired via one of three processes, including non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR).Although NHEJ and MMEJ are recognized as the main repair mechanisms for dsDNA breaks, these pathways, which act by religating broken DNA ends in the absence of a DNA template from an exogenous donor, are error-prone (for review, Xue & Greene, 2021).Indeed, with speci c regard to HIV-1, mutations induced by NHEJ processes facilitated viral escape (Wang G et al., 2016).In sharp contrast, HDR is the preferred repair mechanism for CRISPR/Cas9-induced dsDNA breaks, as it more accurately repairs the genome via a homologous donor template; upregulation of end joining dsDNA repair mechanisms (i.e., NHEJ, MMEJ) in eukaryotic cells, however, limits the extent to which HDR is utilized (Xue & Greene, 2021).Hence, regulating the pathway used to repair dsDNA breaks during genome editing has the potential to enhance the e cacy and precision of CRISPR/Cas9 genome editing.
The utilization of primary mixed glial cultures, rather than puri ed microglia, to evaluate the e cacy of CRISPR/Cas9 is of fundamental importance.Under homeostatic conditions in vivo, the morphology of microglial cells is characterized by a small cell body and very ne, rami ed processes.Puri ed microglia in culture, however, display an amoeboid-like phenotype (Cristóvão et al., 2010, Caldeira et al., 2014) and express a lysosomal enzyme (i.e., CD68) associated with phagocytic activity during the rst ten days in vitro (Cristóvão et al., 2010).Changes to environmental tissue conditions (i.e., in vitro vs. in vivo) also induces pronounced alterations in the transcriptomes and epigenomic features of microglia (Bohlen et al., 2017;Gosselin et al., 2017), whereby gene expression alterations occur in a time-dependent manner (Bohlen et al., 2017).Indeed, serum supplementation, which has historically been utilized to stimulate cellular growth (Puck et al., 1958) and considered fundamental to the viability of cells in culture (Puck et al., 1958), is involved in the perturbation of microglial properties in vitro (Bohlen et al., 2017;Montilla et al., 2020).In light of these ndings, serum-free conditions resembling the physiological condition of cerebrospinal uid have been de ned; conditions that also promote microglial survival and a rami ed microglial morphology in vitro (Bohlen et al., 2017;Montilla et al., 2020).Nevertheless, results derived from only primary microglial cultures should be interpreted with caution, as in vitro models are not without limitation.

Figures Figure 1 Experimental
Figures

Figure 3 E
Figure 3

Figure 4 Even
Figure 4 (Steel et al., 2008)))ndanzo & Chang, 2014) 2014)resembling HIV-1 seropositive individuals on cART.Features of the HIV-1 Tg rat support its utility as a biological system to model neuroHIV (for review,Vigorito et al., 2015)and noninfectious comorbidities(Denaro et al., 2020).Indeed,Moore et al., 2006, Weiss etal., 2021; HIV-1 Tg Rat: Roscoe et al., 2014, McLaurin et al., 2018a, McLaurin et al., 2021), and progressive neurocognitive impairments (Clinical: Heaton et al., 2015, Sacktor et al., 2016, Rubin et al., 2017, Gott et al., 2017; HIV-1 Tg Rat: McLaurin et al., 2016; McLaurin et al., 2019c).Hence, the HIV-1 Tg rat afforded an in vivo biological system with compelling face validity to further evaluate the susceptibility of mixed glia to gene editing via CRISPR/Cas9.HIV-1 Tg rats retro-orbitally inoculated with CRISPR/Cas9 exhibited a pronounced decrease in the number of HIV-1 mRNA in the mPFC relative to their saline-inoculated counterparts.Recombinant adenoassociated viral (rAAV) vectors, which are utilized to deliver exogenous DNA to rodents (e.g., CRISPR/Cas9), can be intravenously delivered via the tail vein(Foust etal., 2009; Gray et al., 2011), facial vein (Foust et al., 2009), or retro-orbital venous sinus (Prabhakar et al., 2019; for protocol, Prabhakar et al., 2021; Present Study).During retro-orbital inoculation, rAAV vectors are slowly injected into the orbital venous plexus, which ows through the superior ophthalmic vein to the cavernous sinus (i.e., part of the brain's dural venous sinuses; Ngnitewe Massa et al., 2022) providing direct access to the CNS.The advantages of retro-orbital, rather than tail vein, injections cannot be understated, as retro-orbital injections are suitable for all ages (e.g., Newborn: Gruntman et al., 2017, Adults: for protocol, Yardeni et al., 2011) and induce a lower stress response(Steel et al., 2008).Indeed, retro-orbital inoculation of HIV-1 Tg rats with CRISPR/Cas9 illustrates the utility of the delivery approach, as well as the susceptibility of mixed glia to gene editing in vivo.McLaurin et al., 2019a) is of fundamental importance, as these analyses suggest that the enhancement of PPI by CRISPR/Cas9 may lead to the mitigation of HIV-1-associated neurocognitive impairments more broadly.Additional studies remain necessary to optimize the CRISPR/Cas9 treatment conditions and evaluate its therapeutic potential to mitigate alterations in higher-order cognitive processes induced by the constitutive expression of HIV-1 viral proteins.Taken together, proof-of-concept studies demonstrate the susceptibility of mixed glia to e cacious gene editing via CRISPR/Cas9 in vitro and in vivo.Fundamental challenges for the complete eradication of the HIV-1 viral genome remain, however, as incomplete excision and signi cant individual variability were observed.Nevertheless, the clinical relevance of CRISPR/Cas9 cannot be understated, as the knockdown of HIV-1 mRNA partially restores the developmental trajectory of temporal processing.Thus, even in the absence of full viral eradication, gene editing via CRISPR/Cas9 may afford a novel therapeutic strategy for HAND.DeclarationsCONFLICT OF INTEREST K.K. is named as an inventor on patents that cover the viral gene editing technology that is the subject of this article.In addition, K.K. is a co-founder, board member (observer), and chief scienti c adviser and hold equity in Excision Biotherapeutics, a biotech startup that has licensed the viral gene editing technology from Temple University for commercial development and clinical trials.All other authors have no interests to disclose.