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Adeno-associated virus (AAV) vectors such as AAV6, which shows tropism for primary human CD4(+) T cells in vitro, are being explored for delivery of anti-HIV therapeutic modalities in vivo. However, pre-existing immunity and sequestration in nontarget organs can significantly hinder their performance. To overcome these challenges, we investigated whether immunosuppression would allow gene delivery by AAV6 or targeted AAV6 derivatives in seropositive rhesus macaques. Animals were immune suppressed with rapamycin before intravenous (IV) or subcutaneous (SC) delivery of AAV, and we monitored vector biodistribution, gene transfer, and safety. Macaques received phosphate-buffered saline, AAV6 alone, or an equal dose of AAV6 and an AAV6-55.2 vector retargeted to CD4 through a direct ankyrin repeat protein (DARPin). AAV6 and AAV6-55.2 vector genomes were found in peripheral blood mononuclear cells and most organs up to 28 days postadministration, with the highest levels seen in liver, spleen, lymph nodes (LNs), and muscle, suggesting that retargeting did not prevent vector sequestration. Despite vector genome detection, gene expression from AAV6-55.2 was not detected in any tissue. SC injection of AAV6 facilitated efficient gene expression in muscle adjacent to the injection site, plus low-level gene expression in spleen, LNs, and liver, whereas gene expression following IV injection of AAV6 was predominantly seen in the spleen. AAV vectors were well tolerated, although elevated liver enzymes were detected in three of four AAV-treated animals 14 days after rapamycin withdrawal. One SC-injected animal had muscle inflammation proximal to the injection site, plus detectable T cell responses against transgene and AAV6 capsid at study finish. Overall, our data suggest that rapamycin treatment may offer a possible strategy to express anti-HIV therapeutics such as broadly neutralizing antibodies from muscle. This study provides important safety and efficacy data that will aid study design for future anti-HIV gene therapies.

Chronic hepatitis B virus (HBV) infection is a major public health problem. New treatment approaches are needed because current treatments do not target covalently closed circular DNA (cccDNA), the template for HBV replication, and rarely clear the virus. We harnessed adeno-associated virus (AAV) vectors and CRISPR-Staphylococcus aureus (Sa)Cas9 to edit the HBV genome in liver-humanized FRG mice chronically infected with HBV and receiving entecavir. Gene editing was detected in livers of five of eight HBV-specific AAV-SaCas9-treated mice, but not control mice, and mice with detectable HBV gene editing showed higher levels of SaCas9 delivery to HBV+ human hepatocytes than those without gene editing. HBV-specific AAV-SaCas9 therapy significantly improved survival of human hepatocytes, showed a trend toward decreasing total liver HBV DNA and cccDNA, and was well tolerated. This work provides evidence for the feasibility and safety of in vivo gene editing for chronic HBV infections, and it suggests that with further optimization, this approach may offer a plausible way to treat or even cure chronic HBV infections., Graphical Abstract, This study analyzes CRISPR-Cas9-based antiviral therapy in a model of chronic hepatitis B virus (HBV) infection. The study demonstrates that therapy is safe, and that gene editing of HBV can occur in vivo, although low levels of CRISPR-Cas9 delivery limit the current efficacy of the approach.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection., Herpes simplex virus establishes lifelong latency in ganglionic neurons, which are the source for recurrent infection. Here Aubert et al. report a promising antiviral therapy based on gene editing with adeno-associated virus-delivered meganucleases, which leads to a significant reduction in ganglionic HSV loads and HSV reactivation.

Objectives A promising curative approach for HIV is to use designer endonucleases that bind and cleave specific target sequences within latent genomes, resulting in mutations that render the virus replication incompetent. We developed a mathematical model to describe the expression and activity of endonucleases delivered to HIV-infected cells using engineered viral vectors in order to guide dose selection and predict therapeutic outcomes. Methods We developed a mechanistic model that predicts the number of transgene copies expressed at a given dose in individual target cells from fluorescence of a reporter gene. We fitted the model to flow cytometry datasets to determine the optimal vector serotype, promoter and dose required to achieve maximum expression. Results We showed that our model provides a more accurate measure of transduction efficiency compared with gating-based methods, which underestimate the percentage of cells expressing reporter genes. We identified that gene expression follows a sigmoid dose-response relationship and that the level of gene expression saturation depends on vector serotype and promoter. We also demonstrated that significant bottlenecks exist at the level of viral uptake and gene expression: only ∼1 in 220 added vectors enter a cell and, of these, depending on the dose and promoter used, between 1 in 15 and 1 in 1500 express transgene. Conclusions Our model provides a quantitative method of dose selection and optimization that can be readily applied to a wide range of other gene therapy applications. Reducing bottlenecks in delivery will be key to reducing the number of doses required for a functional cure.

Background RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. Results We identified a set of gRNAs targeting HIV LTR, gag, and pol using publicly available sequences for these genes and ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A–C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally available sequences. We then selected 59 gRNAs from our list of highly conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon cART cessation even with many doses. Conclusions Target site selection must account for global and within host viral genetic diversity. Globally conserved target sites are good starting points for design, but multiplexing is essential for depleting quasispecies and preventing viral load rebound upon therapy cessation. Electronic supplementary material The online version of this article (10.1186/s12915-018-0544-1) contains supplementary material, which is available to authorized users.

Chronic viral infections remain a major public health issue affecting millions of people worldwide. Highly active antiviral treatments have significantly improved prognosis and infection-related morbidity and mortality, but have failed to eliminate persistent viral forms. Therefore, new strategies to either eradicate or control these viral reservoirs are paramount to allow patients to stop antiretroviral therapy and realize a cure. Viral genome disruption based on gene editing by programmable endonucleases is one promising curative gene therapy approach. Recent findings on RNA-guided Human immunodeficiency virus (HIV)-1 genome cleavage by Cas9 and other gene-editing enzymes in latently infected cells have shown high levels of site-specific genome disruption and potent inhibition of virus replication. However, HIV-1 can readily develop resistance to genome editing at a single antiviral target site. Current data suggests that cellular repair associated with DNA double stranded breaks (DSBs) can accelerate emergence of resistance. On the other hand, a combination antiviral target strategy can exploit the same repair mechanism to functionally cure HIV-1 infection in vitro while avoiding the development of resistance. This perspective summarizes recent findings on the biology of resistance to genome editing, and discusses the significance of viral genetic diversity on application of gene-editing strategies towards cure.

RNA-guided CRISPR/Cas9 systems can be designed to mutate or excise the integrated HIV genome from latently infected cells and have therefore been proposed as a curative approach for HIV. However, most studies to date have focused on molecular clones with ideal target site recognition and do not account for target site variability observed within and between patients. For clinical success and broad applicability, guide RNA (gRNA) selection must account for circulating strain diversity and incorporate the within-host diversity of HIV. To address this, we identified a set of gRNAs targeting HIV LTR, gag and pol using publicly available sequences for these genes. We ranked gRNAs according to global conservation across HIV-1 group M and within subtypes A-C. By considering paired and triplet combinations of gRNAs, we found triplet sets of target sites such that at least one of the gRNAs in the set was present in over 98% of all globally-available sequences. We then selected 59 gRNAs from our list of highly-conserved LTR target sites and evaluated in vitro activity using a loss-of-function LTR-GFP fusion reporter. We achieved efficient GFP knockdown with multiple gRNAs and found clustering of highly active gRNA target sites near the middle of the LTR. Using published deep-sequence data from HIV-infected patients, we found that globally conserved sites also had greater within-host target conservation. Lastly, we developed a mathematical model based on varying distributions of within-host HIV sequence diversity and enzyme efficacy. We used the model to estimate the number of doses required to deplete the latent reservoir and achieve functional cure thresholds. Our modeling results highlight the importance of within-host target site conservation. While increased doses may overcome low target cleavage efficiency, inadequate targeting of rare strains is predicted to lead to rebound upon ART cessation even with many doses.Author summaryThe field of genome engineering has exploded over the last decade with the discovery of targeted endonucleases such as CRISPR/Cas9. Endonucleases are now being used to develop a wide range of therapeutics and their use has expanded into antiviral therapy against latent viral infections like HIV. The idea is to induce mutations in latent viral genomes that will render them replication-incompetent, thereby producing a functional cure. Although a great deal of progress has been made, most studies to date have relied on molecular clones that represent “ideal” targets. For clinical success and broad applicability, these therapies need to account for viral genetic diversity within and between individuals. Our paper examines the impact of HIV diversity on CRISPR-based cure strategies to determine the predictors of future clinical success. We performed an exhaustive and detailed computational analysis to identify optimal CRISPR target sites, taking into consideration both within-host and global viral diversity. We coupled this with laboratory testing of highly-conserved guides and compared measured activity to predicted results. Finally, we developed a mathematical model to predict the impact of enzyme activity and viral diversity on the number of doses of a CRISPR-based therapy needed to achieve a functional cure of HIV.

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials.

The combination of yeast surface display and flow cytometric analyses and selections is being used with increasing frequency to alter specificity of macromolecular recognition, including both protein-protein and protein-nucleic acid interactions. Here we describe the use of yeast surface display and cleavage-dependent flow cytometric assays to increase the specificity of an engineered meganuclease. The re-engineered meganuclease displays a significantly tightened specificity profile, while binding its cognate target site with a slightly lower, but still sub-nanomolar affinity. When incorporated into otherwise identical megaTAL protein scaffolds, these two nucleases display significantly different activity and toxicity profiles in cellulo. The structural basis for reprogrammed DNA cleavage specificity was further examined via high-resolution X-ray crystal structures of both enzymes. This analysis illustrated the altered protein-DNA contacts produced by mutagenesis and selection, that resulted both in altered readout of those based and a necessary reduction in DNA binding affinity that were necessary to improve specificity across the target site. The results of this study provide an illustrative example of the potential (and the challenges) associated with the use of surface display and flow cytometry for the retargeting and optimization of enzymes that act on nucleic acid substrates in a sequence-specific manner.

A large portion of the global population carries latent herpes simplex virus (HSV), which can periodically reactivate, resulting in asymptomatic shedding or formation of ulcerative lesions. Current anti-HSV drugs do not eliminate latent virus from sensory neurons where HSV resides, and therefore do not eliminate the risk of transmission or recurrent disease. Here, we report the ability of HSV-specific endonucleases to induce mutations of essential HSV genes both in cultured neurons and in latently infected mice. In neurons, viral genomes are susceptible to endonuclease-mediated mutagenesis, regardless of the time of treatment after HSV infection, suggesting that both HSV lytic and latent forms can be targeted. Mutagenesis frequency after endonuclease exposure can be increased nearly 2-fold by treatment with a histone deacetylase (HDAC) inhibitor. Using a mouse model of latent HSV infection, we demonstrate that a targeted endonuclease can be delivered to viral latency sites via an adeno-associated virus (AAV) vector, where it is able to induce mutation of latent HSV genomes. These data provide the first proof-of-principle to our knowledge for the use of a targeted endonuclease as an antiviral agent to treat an established latent viral infection in vivo.

Highly active antiretroviral treatment has dramatically improved the prognosis for people living with HIV by preventing AIDS-related morbidity and mortality through profound suppression of viral replication. However, a long-lived viral reservoir persists in latently infected cells that harbor replication-competent HIV genomes. If therapy is discontinued, latently infected memory cells inevitably reactivate and produce infectious virus, resulting in viral rebound. The reservoir is the biggest obstacle to a cure of HIV.This review summarizes significant advances of the past year in the development of cellular and gene therapies for HIV cure. In particular, we highlight work done on suppression or disruption of HIV coreceptors, vectored delivery of antibodies and antibody-like molecules, T-cell therapies and HIV genome disruption.Several recent advancements in cellular and gene therapies have emerged at the forefront of HIV cure research, potentially having broad implications for the future of HIV treatment.

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One newly emerging approach to cure persistent viral infections is via the use of targeted endonucleases. To date, endonucleases have been used as therapeutic agents targeting the genomes of viruses including EBV, JCV, HBV, HCV, HIV, HPV, HSV and HTLV, and have been shown to inhibit viral replication and subsequently persistence. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of a panel of four HIV pol-specific zinc finger nucleases (ZFNs) we identified a provirus encoding a treatment-resistant and infectious mutant virus that was derived from a ZFN2-mediated disruption of reverse transcriptase (RT). Although ZFN-mediated disruption of HIV protease (ZFN1), RT (ZFN2 & ZFN3) and integrase (ZFN4) coding sequences could inhibit viral replication, a RT mutant provirus was detected that produced a replication competent and ZFN2 cleavage resistant HIV. Mutant virus ZFN2(+3) contained a single amino acid insertion that introduced a Leucine-Leucine (LL) motif in the thumb domain of RT. The RT of mutant virus ZFN2(+3) likely remained functional since a LL motif is also found in the RT of several hominid, old world monkey or equine spumaviruses. In ZFN2-treated HEK293 and CD4+ SupT1 T cells, we found that up to 25% and 6% of all ZFN2 HIV pol target site mutations detected by Illumina sequencing encoded the ZFN2(+3) mutant respectively. We found that ZFN2(+3) mutant virus could replicate at levels comparable to wild type HIV in primary CD4+ T cells, but importantly remained susceptible to treatment with both NRTi and NNRTi antiretroviral inhibitors. When secondary ZFN-derived mutations were introduced into other RT or integrase domains of mutant virus ZFN2(+3), replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.