Other cure related studies at CROI 2019
The following reports are taken from a longer article that also includes coverage of the two new cases of HIV remission off ART that we reported in an earlier issue of HTB.
Gene therapy: SB-728-T
Outside of the setting of stem cell transplants for cancers, the leading approach to knocking out CCR5 from CD4 T cells has been Sangamo Therapeutics SB-728-T. In clinical trials, CD4 T cells are extracted from individuals with HIV, edited at the CCR5 gene using zinc finger nuclease technology, and then expanded and reinfused. The company was hoping to achieve control of HIV viral load after ART interruption, but so far it hasn’t proven possible to modify sufficient numbers of CD4 T cells. Further commercial development for HIV has been abandoned, but some investigator-initiated studies continue.
Pablo Tebas presented new results from a trial of SB-728-T that broadly conformed to previous research. 
A total of 14 individuals on ART received a single infusion of modified CD4 T cells, either with or without a preceding dose of cyclophosphamide (intended to deplete existing CD4 T cells and make more room for modified cells). In a slight wrinkle, the delivery of the zinc finger nucleases to the cells was achieved using messenger RNA instead of the adenovirus vector employed in prior studies; the proportion of CD4 T cells successfully edited at the CCR5 gene by the two approaches was similar. The protocol included an ART interruption, and Tebas noted that there was a slight delay in viral load rebound compared to historical controls, but no cases of prolonged containment of HIV.
As observed in prior trials, participants heterozygous for the CCR5Δ32 mutation appeared to respond best. Because these individuals already have one disabled CCR5 gene, the zinc finger nucleases only have to edit one of the two alleles present in each CD4 T cell in order to prevent expression of a functional CCR5 co-receptor. Tebas concluded that more efficient CCR5 modification could potentially lead to more stringent control of HIV off ART, but it appears unlikely that such an outcome can be achieved with SB-728-T.
One variation on the theme of trying to genetically protect CD4 T cells from HIV infection involves focusing on modifying cells capable of recognising and responding to the virus (HIV-specific CD4 T cells). A seminal study by Danny Douek many years ago showed that the virus preferentially infects HIV-specific CD4 T cells, which become dysfunctional and unable to perform their task of coordinating an effective immune response to the virus. 
The company American Gene Technologies is pursuing a strategy involving the genetic modification of HIV-specific CD4 T cells, with trials planned soon, and results should shed light on whether this is a better approach than attempting to modify CD4 T cells in bulk. 
Taking a bite out of the latent reservoir with CRISPR/Cas9
One of the more intuitively appealing ideas in cure research involves attempting to cut the integrated HIV genome out of the DNA of latently infected cells. In this scenario, gene-editing strategies are targeted against the virus itself rather than a host gene like CCR5. The goal is to perform a sort of genetic surgery, excising HIV genes from infected cells without damaging the cell’s genome.
The gene-editing tool CRISPR/Cas9 has emerged as the leading candidate in this research, and some very preliminary results in mouse models have suggested it may have potential.  The laboratory of Kamel Khalili at Temple University has pioneered these studies, in tandem with Excision Biotherapeutics, a company Khalili founded to move the approach into the clinic.
At CROI 2019, Tricia Burdo from Temple University debuted the results of a study exploring whether CRISPR/Cas9 could excise latent SIV in the SIV/macaque model of HIV infection. 
The cutting machinery of CRISPR/Cas9 is aimed at a target by the inclusion of molecules called guide RNAs (gRNAs), and in this case the researchers created gRNAs capable of recognising three relatively conserved sites in the SIV genome (two in the long terminal repeats present at either end of the genome, and one in the gag gene). Burdo noted that the targeting of multiple sites is necessary for both attempting to excise large chunks of the viral genome and avoiding the potential development of resistance (somewhat similar to the rationale for combination ART). The technique produces “very little to no off-target effects,” according to Burdo.
The SIV-targeted CRISPR/Cas9 was delivered using an adeno-associated virus serotype nine (AAV9) vector. AAVs are a popular gene therapy delivery vehicle that can carry their payload into a broad range of both dividing and non-dividing cells without apparent safety issues (two AAV-delivered gene therapies have been approved by regulatory agencies).
The study included three macaques, all infected with SIVmac239 and placed on a suppressive ART regimen. In initial experiments, peripheral blood mononuclear cells (PBMC) sampled from the animals were transduced with the AAV9-CRISPR construct, producing evidence of excision of SIV genes between targeted sites.
AAV9-CRISPR was then infused into two of the macaques at a dose of 1013(ten trillion) copies per kilogram, a lengthy process involving the delivery of 100 mL at a rate of 1 mL per minute. Three weeks after the infusion, animals were euthanised and necropsy studies conducted. The third animal served as a control and was also euthanised to facilitate comparisons with the AAV9-CRISPR recipients.
Burdo reported that prior to euthanasia, fragments of the SIV genome that had been cut at targeted sites—referred to as excision products – could be detected in PBMC from the treated animals, as was observed when PBMC were exposed to AAV9-CRISPR in a laboratory dish. Cas9 DNA could also be detected in cells, indicating uptake of the gene-editing tool.
Necropsy studies included a preliminary evaluation of SIV outgrowth from PBMC samples. The PBMC were combined with SIV-susceptible CEM cells and then SIV p27 Gag protein levels were measured over time. SIV replication could be detected in samples from the control but not those from the macaques that received AAV9-CRISPR. However, Burdo emphasised that this assessment did not involve activating the PBMC to induce virus production (as is the case with the standard virus outgrowth assays used in human studies) – those experiments are pending.
Analyses of Cas9 DNA demonstrated widespread distribution in the tissues of the two treated macaques, ranging from around 1-10 thousand copies per million cells in the brain to over 10 million copies per million cells in the spleen and liver (presumably reflecting the presence of multiple copies in some cells). Burdo also showed evidence of SIV excision products in spleen, lung and several lymph nodes (including inguinal, submandibular, bronchial and colonic) from the animals.
The data appear very encouraging, but do not provide information on the magnitude of effect on the latent SIV reservoir (i.e. exactly how much latent SIV was successfully excised or disabled). In response to a question, Burdo reported that future plans include conducting analytical treatment interruptions in macaques treated with AAV9-CRISPR to assess whether viral load rebound is limited or prevented by the intervention.
An issue not covered in the presentation is the potential for the induction of immune responses against Cas9, which has been observed in mouse studies. 
Because Cas9 is derived from bacteria, it is treated as foreign by the immune system, and AAV vectors can have an adjuvant effect that seems to promote immune responses against AAV-delivered proteins (this has occurred in studiesusing AAV to deliver anti-HIV broadly neutralising antibodies).  Pre-existing anti-Cas9 immune responses have also been detected in humansdue to infection with Staphylococcus aureus and Streptococcus pyogenes. 
In a graph displaying longitudinal viral load measurements in the macaques (on slide #4 in the webcast), it looks as if administration of AAV9-CRISPR may have been temporally associated with a transient increase in SIV viral load – which could be suggestive of immune activation – although there were also viral load fluctuations in other animals. Gaining an understanding of whether AAV9-CRISPR delivery can activate the immune system and lead to the generation (or activation) of anti-Cas9 immune responses will be important prior to initiating human trials.
A theoretical concern that researchers have raised about strategies aiming to excise latent HIV relates to what might occur in cells that have more than one integrated copy of the HIV genome (this phenomenon is thought to be uncommon, but has been reported). [9, 10]
In this situation, it’s possible that rather than just removing HIV genes, an excision approach might make cuts in each of the separate integrated virus genomes and thereby remove all of the cell’s DNA located between the different HIV integration sites. Damaging the genome of a cell in this way could potentially have untoward effects.
Overall, Burdo’s results offer significant encouragement for efforts to translate the approach into human clinical trials. Kamel Khalili and colleagues are now working toward that goal in collaboration with Jeffrey Jacobson, a highly experienced clinical HIV researcher who joined Temple University in 2016.
Attack of the repliclones
The past few years have seen an increasing focus on the role of CD4 T cell proliferation in sustaining the latent HIV reservoir. Evidence has accumulated demonstrating that HIV proviruses can be faithfully copied into the daughter cells of latently infected CD4 T cells when they proliferate—the phenomenon can be discerned by the detection of genetically matching copies of the HIV provirus integrated into the exact same place in the genome of multiple CD4 T cells (the progeny of proliferating CD4 T cells are known as clones). Mathematical modeling suggests CD4 T cell proliferation may be the primary mechanism that allows the latent HIV reservoir to persist, and decline only very slowly over time. 
Elias Halvas from the University of Pittsburgh showed at CROI 2019 that CD4 T cell clones containing integrated, intact HIV DNA are a source of low-level HIV viral load that can be detected in some individuals on ART. Essentially, some of these cells can spit out sufficient amounts of HIV RNA to be detectable, even though the virus is not actually replicating (i.e. going on to infect other cells—this is prevented by ART). 
Halvas’s study involved 10 people who had been referred due to persistent low-level viral load despite ART (HIV RNA >20 copies/mL occurring for at least 6 months). The average time on treatment was 10 years, and viral load ranged from 40 to 356 copies/mL, with a median of 97.5 copies/mL.
One individual displayed evidence of ongoing HIV evolution and the development of drug resistance mutations and was considered a case of ART regimen failure, excluding them from further analysis.
Samples from the remaining nine showed the presence of genetically identical HIV RNA at multiple timepoints, and there was no sign of viral evolution or resistance mutations against current ART. The source of the HIV RNA was identified as CD4 T cell clones containing integrated, replication-competent HIV DNA (Halvas has christened them “repliclones”). In four cases the genetic sequence of the HIV RNA could be matched to viruses detected in the quantitative virus outgrowth assay (qVOA).
Halvas concluded that the possibility of production of HIV RNA by infected CD4 T cell clones needs to be borne in mind by clinicians caring for people with HIV, who might otherwise suspect that persistently detectable low-level viral load indicated non-adherence or treatment failure.
As to the implications for HIV cure research, Halvas suggested that repliclones may contribute to rapid viral load rebound after ART interruption (when the HIV RNA they produce is able to start infecting other cells), and he stressed that they will need to be targeted for elimination or suppression. The mechanisms prompting HIV RNA production by the cells are unclear, and need to be elucidated.
Notably, the data indicate that HIV latency can be more dynamic than was initially appreciated. It’s now clear that in some CD4 T cell clones containing integrated HIV DNA, the virus is not permanently latent, because there are times when production of HIV RNA is detectable.
In an article by Jon Cohen for Science that covers the study, John Mellors points out that the data raise questions about the “kick & kill” strategy in HIV cure research. 
The rationale for providing a latency-reversing “kick” is that most latently infected cells do not produce HIV RNA and therefore remain invisible to the immune system. Halvas’s results demonstrate that at least some latently infected cells do intermittently generate HIV RNA, and don’t die off as a result.
One salutary possibility is that researchers developing “kill” strategies may be able to study their efficacy in individuals like those described by Havlas, who already have low-level viral load on ART without the need for administration of any latency-reversing candidate. In theory, an effective “kill” approach should be able to reduce the amount of HIV RNA detected in such cases.
Targeting the latent HIV reservoir with anti-proliferative therapy
The recognition that the HIV reservoir is at least partly sustained by the proliferation of CD4 T cells is rekindling interest in testing the effects of anti-proliferative therapies in the context of cure research.
At the pre-CROI community HIV cure research workshop, Joshua Schiffer from the Fred Hutchinson Cancer Research Center described a small (four person) pilot trial of the anti-proliferative drug mycophenolate mofetil (MMF) being conducted by his research group with funding from amfAR. The rationale is based on the results of mathematical modeling work suggesting that inhibiting CD4 T cell proliferation in people on ART should significantly accelerate the decay of the HIV reservoir. Results are anticipated to be available for CROI 2020. 
Links to the video of Joshua Schiffer’s talk are on the workshop web page, along with the slides. 
Timothy Heinrich from UCSF presented results of an AIDS Clinical Trials Group (ACTG) trial of sirolimus (a drug with potent anti-proliferative activity, also known as rapamycin) in people on suppressive ART. In the 16 participants who completed 20 weeks of dosing, there was a slight but statistically significant 0.16 logs reduction in HIV DNA levels. CD4 T cell expression of the proliferation marker Ki67 was also significantly reduced. 
Heinrich noted that rates of sirolimus discontinuation were high, and there were also transient increases in the inflammatory biomarkers IL-6 and sCD14 and the coagulation biomarker D-Dimer. The results appear consistent with the notion that inhibiting CD4 T cell proliferation can affect HIV reservoir size, but additional research is needed to confirm that this was the primary mechanism for the HIV DNA reduction.
One of the pioneers in this area of HIV cure research is Andrea Savarino, who reported many years ago that the gold-based anti-proliferative drug auranofin reduced the SIV reservoir in ART-treated macaques. 
Since that time, Savarino and colleagues have collaborated with investigators in Brazil to conduct a small pilot trial involving auranofin (which is a licensed treatment for rheumatoid arthritis). The latest results were presented in a poster at CROI 2019. 
The study design is complicated, involving multiple interventions administered to six groups, each with just five participants. The researchers report that auranofin combined with several other agents led to a reduction in HIV DNA, but the contribution of the anti-proliferative effect is unclear. Administration of the drug was not associated with any serious side effects. Given the renewed interest in targeting CD4 T cell proliferation and the uncertain safety profile of some anti-proliferative drugs, additional studies of auranofin may be justified.
Jefferys R. TAG BSVC Blog. (11 April 2019).
Unless stated otherwise, references are to the programme and abstracts of the Conference on Retroviruses and Opportunistic Infections, 4–7 March 2019, Seattle.
- Tebas P et al. Delayed viral rebound during ATI after infusion of CCR5 ZFN-treated CD4 T cells. CROI 2019, Seattle. Oral abstract 25.
- Douek D et al. HIV preferentially infects HIV-specific CD4+ T cells. Nature 417;95-98 (2002).
- American Gene Technologies.
- Bella R et al. Removal of HIV DNA by CRISPR from patient blood engrafts in humanized mice. Molecular Therapy 12:275-282, doi: 10.1016/j.omtn.2018.05.021
- Burdo T et al. Ex vivo and in vivo editing of the SIV genome in nonhuman primates by CRISPR-CAS9. CROI 2019, Seattle. Oral abstract 25.
- Wang D et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum Gene Ther. 2015 Jul 1; 26(7): 432–442.
- Priddy FH et al. Adeno-associated virus vectored immunoprophylaxis to prevent HIV in healthy adults: a phase 1 randomised controlled trial. Lancet HIV, 6(4);PE230-E239. (01 April 2019).
- Charlesworth CT et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nature Medicine 25;249-254. (2019).
- Karpinski J et al. Directed evolution of a recombinase that excises the provirus of most HIV-1 primary isolates with high specificity. Nature Biotechnology34;401-409 (2016).
- Josefsson L et al. Majority of CD4+ T cells from peripheral blood of HIV-1–infected individuals contain only one HIV DNA molecule. Proc Natl Acad Sci USA. 2011, 108(27):11199–11204.
- Reeves DB et al. A majority of HIV persistence during antiretroviral therapy is due to infected cell proliferation. Nature Communications vol9: 4811 (2018).
- Halvas E et al. Nonsuppressible viremia on ART from large cell clones carrying intact proviruses. CROI 2019, Seattle. Oral abstract 23.
- Cohen J. Curing HIV just got more complicated. Can CRISPR help? Science (15 March 2019).
- Reeves DB et al. Anti-proliferative therapy for HIV cure: a compound interest approach. Scientific Reports. 7: 4011 (2017).
- TAG.Pre-CROI Community HIV Cure Research Workshop 2019.
- Henrich T et al. Sirolimus reduces T-cell cycling and immune checkpoint marker expression, ACTG A5337. CROI 2019, Seattle. Oral abstract 131.
- Savarino A et al. Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension. AIDS 25(11):1347–1356. doi: 10.1097/QAD.0b013e328347bd77. (17 July 2011).
- Diaz RH et al. Randomized trial of impact of multiple interventions on HIV reservoir: SPARC-7 trial. CROI 2019, Seattle. Poster abstract 399.