**5. Genome editing**

The ability to engineer specific changes in the genome of an organism has developed rapidly over the last 10 years. The technology of gene editing relies on nucleases, scissor-like enzymes, with the ability to cut genomic DNA in a highly specific manner. This process results in additions, deletions or alterations at the targeted site of the genome. There are two pathways that can achieve double stranded breaks (DSB) in DNA; (i) nonhomologous end joining (NHEJ) repair pathway, where deletions or insertions in the target gene result in gene disruption *e.g.* CCR5, or (ii) the homologous recombination or homology-directed repair (HDR) pathway, in which DNA sequences are introduced into the genome using a homologous DNA template. The HDR pathway is more precise, with limited off-target genome effects, due to more control over the integration site, copy number and expression of the DNA sequence [73]. Some examples of gene editing technologies include zinc finger nuclease (ZFN), transcription activator-like nucleases (TALENS), clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Examples of specific gene editing technologies that have been explored in the context of HIV treatment are described below.

#### **5.1. Zinc finger nucleases (ZFN)**

ZFNs are a combination of zinc-finger proteins, which have DNA recognition specificity, and the nuclease activity of the cleavage domain of restriction enzyme, *Fok*I. The most advanced ZFN pair targets the CCR5 host gene and HIV-1 co-receptor [74]. Preclinical studies in a mouse model, where mice received CD3/CD28-activated primary CD4+ T cells treated with the ZFN delivered in a chimeric adenoviral vector, Ad5/F35, showed anti-HIV efficacy, with disruption of 40–60% of all CCR5 alleles and 33% disruption of both CCR5 alleles [74]. Following the successful pre-clinical data, the ZFN progressed to phase I clinical studies and the first-in-human gene editing HIV treatment trial (#NCT00842634) commenced in 2009.

The primary outcome of this study investigated the safety of ZFN modification of autologous CD4+ T cells being delivered to HIV positive individuals [75]. A secondary outcome measured immune reconstitution and HIV resistance. Twelve ART-treated patients with undetectable viral loads were enrolled in two cohorts dependent on CD4+ T cell count; cohort 1 included patients with CD4+ T cell count >450/mm3 , the median being 662/mm3 , and cohort 2 was patients with lower CD4+ T cells counts between 200 and 500/mm3 , the median being 272/mm3 [75]. Patients received one infusion containing 5x107 autologous CD4+ T cells that were ZFN-modified. The infusion of ZFN-treated cells was deemed safe, with one serious adverse event reported that was infusion-related. All patients demonstrated engraftment, with the ZFN-modified cells being present for up to ≥42 months following infusion and showed expected characteristics. At 4 weeks post-infusion, cohort 1 ceased taking ART in a 12 week analytical treatment interruption (ATI), resulting in four out of six patients with detectable viral loads at 2–4 weeks post-ART cessation [75]. One of the six patients experienced a delayed increase in viral load at week 6, but was still below the viral set point [75]. This patient was later determined to be heterozygous for CCR5Δ32, suggesting that this genotype enhanced the ZFN treatment effect.

The successful modification of CD34+ HPSCs was also shown using the same ZFN pair [76]. This has been further optimised to achieve HDR-induced gene modifications using an adenoassociated virus vector (AAV) serotype 6 and electroporation to deliver nuclease mRNA to both primary CD4+ T cells and HPSCs [77], achieving between 8 and 60% and 15–40% CCR5 editing, respectively. Currently, there is no *in vivo* method that can effectively deliver nucleases to cells infected with HIV, and this will require further characterisation of the HIV sanctuary sites and identification of latent cell markers to allow specific targeting of cells that comprise the virus reservoir.

The generation of off-target genome modification is also a concern for the clinical application of ZFNs. This is exemplified by the off-target effect reported for the highly related CCR2 gene which was disrupted in 5.39% of ZFN-modified CD4+ T cells that were targeting CCR5 and decreased CCR5 expression by 36% [74]. Optimisation of the CCR5 ZFN would be required if this off-target effect was determined to be deleterious.

#### **5.2. CRISPR/Cas9**

this case may be limited due to the patient recently commencing pre-exposure prophylaxis (PrEP) in order to prevent contracting HIV a second time. Firstly, the patient had ceased taking ART for >4 years without experiencing viral rebound, secondly, the viral DNA level was below detection limit in the periphery and in tissue biopsies, and thirdly, the patient showed a decrease in anti-HIV antibodies, all indicating a lack of virus replication, which makes it possible to conclude that the patient is functionally cured of HIV [66, 71]. The principle of a sterilising cure is the complete eradication of a pathogen out of the human body. This would therefore mean that every single cell previously infected and therefore carrying the HIV-1 genome would need to be replaced by new donor-derived cells to completely eradicate HIV from the body. All tests for proviral DNA until now, showed no detectable HIV-DNA and Timothy Ray Brown remained without viral rebound for 4 years, indicating the possibility that even the long-lasting memory immune cells were replaced by cells derived from the donor. This could lead to the interpretation that it was in fact a sterilising cure. That being said it is important to take into account the current limits of detection and the fact every single cell in his body cannot be analysed. Further similar results were found in two other patients who did eventually rebound. Therefore, one cannot be definitive in whether the cure is functional or completely sterilising. Regardless of whether the final conclusion is potentially a sterilising

The ability to engineer specific changes in the genome of an organism has developed rapidly over the last 10 years. The technology of gene editing relies on nucleases, scissor-like enzymes, with the ability to cut genomic DNA in a highly specific manner. This process results in additions, deletions or alterations at the targeted site of the genome. There are two pathways that can achieve double stranded breaks (DSB) in DNA; (i) nonhomologous end joining (NHEJ) repair pathway, where deletions or insertions in the target gene result in gene disruption *e.g.* CCR5, or (ii) the homologous recombination or homology-directed repair (HDR) pathway, in which DNA sequences are introduced into the genome using a homologous DNA template. The HDR pathway is more precise, with limited off-target genome effects, due to more control over the integration site, copy number and expression of the DNA sequence [73]. Some examples of gene editing technologies include zinc finger nuclease (ZFN), transcription activator-like nucleases (TALENS), clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Examples of specific gene editing technolo-

gies that have been explored in the context of HIV treatment are described below.

ZFNs are a combination of zinc-finger proteins, which have DNA recognition specificity, and the nuclease activity of the cleavage domain of restriction enzyme, *Fok*I. The most advanced ZFN pair targets the CCR5 host gene and HIV-1 co-receptor [74]. Preclinical studies in a mouse model, where mice received CD3/CD28-activated primary CD4+ T cells treated with the ZFN delivered in a chimeric adenoviral vector, Ad5/F35, showed anti-HIV efficacy,

cure, it was derived from a functional cure approach.

52 In Vivo and Ex Vivo Gene Therapy for Inherited and Non-Inherited Disorders

**5. Genome editing**

**5.1. Zinc finger nucleases (ZFN)**

The development of therapeutics using CRISPR/Cas9 technology has rapidly intensified over the last decade. The system is based on a short guide RNA (gRNA) that targets a specific DNA sequence and the Cas9 endonuclease, which then cleaves the double stranded DNA. Mutations, either deletions or insertions, are introduced into the target sequence following DNA repair by the NHEJ repair pathway. Similar to ART, HIV has been shown to develop virus escape mutations when only a single gRNA is utilised and requires multiple gRNAs to prevent the emergences of virus resistance [78, 79]. Multiple studies have reported HIV-1 inactivation using this gene editing platform with dual gRNAs, via mutation at either target sites or complete excision of the virus sequence between the two target sites [80–83]. Although CRISPR technology has proven successful in inactivating HIV-1, like the ZFN system, successful delivery to all cells of the latent reservoir will be challenging without a known latency marker.

widely accepted method for delivering viral vectors to large numbers of cells, in particular to

Mechanisms for Controlling HIV-1 Infection: A Gene Therapy Approach

http://dx.doi.org/10.5772/intechopen.79669

As HSC predominantly reside in the bone marrow, in order to increase the quantity of HSC in peripheral blood, it is common to use granulocyte colony stimulating factor (G-CSF) as a mobilising agent to encourage recirculation of HSC. This causes cells to migrate from the bone marrow and lymph tissue into the peripheral blood. The use of G-CSF or other stimulating factors is essential when HSC are to be transduced with the therapeutic gene/vector, with various trials showing that HSC cell counts in peripheral blood increase 20–50-fold over the course of G-CSF administration [91–93]. To aid with re-engraftment of HSC back into the bone marrow after transduction, a technique known as myeloablation has been utilised in some clinical trials prior to the reintroduction of HSC via infusion, in order to provide an immunological niche and improve engraftment of the gene-containing cells [94]. This procedure involves the eradication of resident HSC, thereby reducing the population of nontransduced cells, and creating more space for the transduced cell population to reconstitute the bone marrow. A delay of the presence of newly 'protected CD4+ T cell' population would

**Figure 4.** Gene therapy delivery strategies; *ex vivo* lentivirus transduction of isolated patient haematoepoetic stem cells (HSC) and/or CD4+ T cells to deliver the gene modification versus systemic, *in vivo* delivery of the gene therapy directly

to the patient, which requires a cell specific moiety to ensure targeted cell delivery.

HSC in the bone marrow [85]. The *ex vivo* gene therapy process is depicted in **Figure 4**.

The CRISPR/Cas9 platform has recently been adapted to activate HIV transcription, akin to the "shock and kill" approach. This involves a mutation in the Cas9 catalytic domain which results in deactivated Cas9 (dCas9). The dCas9 can then be coupled to a strong transcription activation domain (AD) and targeted to the HIV-1 LTR can induce transcriptional activation via recruitment of transcription and chromatin modifying factors. One example of the dCas9-AD system is dCas9-VP64, which contains multiple copies of the herpes simplex virus (HSV) VP16-drived minimal AD and has been shown to activate HIV-1 promoter-driven gene expression. Interestingly, the most promising gRNA target in the HIV-1 5'LTR is single guide (sg)362F [84] and similar to the siPromA sequence described in the "block and lock" approach, targets the NF-κB binding motif.
