**4. Successful gene therapy treatment of HIV-1: the "Berlin patient"**

The first person cured of HIV-1 was Timothy Ray Brown, also known as the 'Berlin patient', who still today remains to be the only person to be cured of HIV-1. Diagnosed with HIV at the age of 29, the patient commenced ART [65], but then presented with acute myeloid leukaemia at the age of 40. At that stage the patient's HIV was controlled with ART and classified as stage A2; asymptomatic with a CD4+ T-cell count of 415 cells/μL [66]. ART interruption during the first initial treatment showed viral rebound, therefore ART was resumed and no further treatment was required until an acute myeloid leukaemia relapse 7 month after initial treatment [66]. The patient then received an allogeneic haematopoietic stem-cell transplantation (HSCT) [65, 66]. HSCT was already shown to be feasible in HIV positive patients, but it was also known that HSCT alone was insufficient to eliminate HIV [67]. For many patients finding an HLA-matched stem-cell donor is a significant challenge, however a suitable match was identified for the Berlin patient and subsequent screens for possible donors with the homozygous CCR5-delta32 (CCR5Δ32/Δ32) allele were performed [65, 66]. High resistance against HIV infection has been reported for individuals who are homozygous for the CCR5-delta32 deletion [68, 69]. HIV requires CD4 and typically either CCR5 (or CXCR4) for cell entry, making it a promising candidate for intervention [69]. Unlike CD4 and CXCR4, the absence of CCR5 is not obviously deleterious for modified cells [69]. Therefore the approach to use CCR5-delta32 stem-cells for HSCT of HIV infected patients was pursued, as earlier described by Chow et al., in 2001 [70]. Using this treatment approach an HLA-matched stem-cell donor with the homozygous CCR5-delta32 allele was identified [66].

HIV-1JR-FL. In human PBMCs that were stably transduced with shPromA delivered by a lentivirus vector and transplanted into the NOJ mice, followed by immune reconstitution, mice were protected from HIV-1 challenge, with significantly decreased plasma viral loads and normal CD4:CD8 T cell ratios, compared to control group treated with cells transduced with an inactive siRNA sequence carrying three mismatches (shPromA-M2) [58]. We anticipate, much like combined ART, that a multiplexed approach of combining TGS-inducing siRNAs will be necessary to provide sufficient control across a wide range of HIV subtypes and strains. The Chattopadhyay laboratory has also reported a TGS-inducing siRNA sequence specifically targeting the HIV-1 subtype C NF-κB triple repeat motif, termed S4-siRNA. They demonstrated S4-siRNA induced TGS in a TZM-bl cell line and *ex vivo* human PBMCs transfected with S4-siRNA and infected with various subtype C viruses, as determined by measuring viral RNA levels [62]. Further, ChIP assay confirmed the enrichment of epigenetic repressive marks using histone methylation markers, H3K27me3 and H3K9me2. This siRNA may have potential as an RNA therapeutic, since HIV-1 subtype C is prevalent in approximately half of

The Morris laboratory has also described a TGS-inducing siRNA, termed, LTR362, which also targets the NF-κB tandem repeat motif [63] and overlaps with 8 bp of the siPromA sequence. This RNA therapeutic has recently been further developed with the addition of a delivery aptamer designed to the HIV-1 glycoprotein termed gp120 A-1 and multiplexing with PTGSinducing siRNAs targeting Tat and Rev. [64], designed by the Rossi laboratory. They showed the LTR362 RNA localised to the nucleus of an HIV-infected T lyphoblastoid CEM cell line and primary human CD4+ T cells. Virus suppression showed a 10-fold reduction of viral p24 levels compared to control cultures at 12 days post-infection. This potential dual therapeutic was assessed *in vivo* using an HIV-1 infected humanised NOD/SCID/IL2 rγnull mouse model and demonstrated suppressed virus infection and protected CD4+ T cell levels in viremic mice. However, the mechanism of virus suppression was determined to be PTGS, due to the lack of the CpG methylation, an epigenetic silencing mark, at the 5'LTR. Investigation of histone methylation may prove some involvement of TGS, however the study currently indicates that while cell-type specific aptamer delivery of TGS-inducing siRNA functions *in vitro*, the *in vivo* silencing effect will require significant optimising to achieve robust epigenetic modifications [64].

**4. Successful gene therapy treatment of HIV-1: the "Berlin patient"**

The first person cured of HIV-1 was Timothy Ray Brown, also known as the 'Berlin patient', who still today remains to be the only person to be cured of HIV-1. Diagnosed with HIV at the age of 29, the patient commenced ART [65], but then presented with acute myeloid leukaemia at the age of 40. At that stage the patient's HIV was controlled with ART and classified as stage A2; asymptomatic with a CD4+ T-cell count of 415 cells/μL [66]. ART interruption during the first initial treatment showed viral rebound, therefore ART was resumed and no further treatment was required until an acute myeloid leukaemia relapse 7 month after initial treatment [66]. The patient then received an allogeneic haematopoietic stem-cell transplantation (HSCT)

the people living with HIV globally.

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

*3.2.2.2. RNA-aptamer silencing*

The patient ceased ART medication on the day prior to the HSCT procedure, which was successful with complete chimerism achieved and only grade I graft-versus-host disease (GvHD) as serious complications [66]. HIV infection was analysed by RNA and DNA-PCR and remained undetectable in peripheral blood and bone marrow, as well as in the rectal mucosa [66]. Analysis of macrophages in the intestinal mucosa found they were still expressing CCR5, indicating that 159 days post-HSCT these long-lasting cells were not yet replaced by the new immune system [66]. The CD4+ T-cell count in peripheral blood stayed at a low level of less than 300 cells/μL after the first HSCT until leukaemia relapsed on day 332 after HSCT [66, 71]. Following a total body irradiation, the patient received a second HSCT from the same CCR5Δ32/Δ32 donor [66, 71]. Fortunately, after the second transplantation the HIV load remained undetectable for the following years in peripheral blood, bone marrow and tissue biopsies, including gut and brain [66, 71]. CCR5-expressing macrophages in the gut became undetectable over the years and the peripheral CD4+ T-cell count increased greatly within the first 6 month after the second HSCT, to over 400 cells/μL [71]. While the treatment was successful in inducing remission from the acute myeloid leukaemia, recovery from the second HSCT was slow, with a long period of infections, GvHD reactions in the liver and a period of fever, dizziness and delirium [65, 71]. The patient experienced loss of short-term memory, was almost paralysed and had to learn to walk again [65, 71].

As a milestone in HIV cure research, there is the question if this is a one-time wonder cure or if it is reproducible? In 2014, Hutter et al. assessed six more cases of patients with HIV-1 receiving an allogenic CCR5Δ32/Δ32 HSCT [72]. Five of those patients died within the first 4 months due to relapse, GvHD or infection [72]. The only patient surviving for 12 months experienced a rebound of CXCR4-tropic HIV-1 rapidly after the transplantation and died from a relapse of cancer [72]. This shows the difficulties of HSCT in HIV infected patients and the importance for careful selection of donor to recipient, as well as considering the continuance of ART to prevent CXCR4-tropic HIV-1 from rebounding until the new immune system has become more established [72]. In light of these attempts to replicate the successful treatment of Timothy Ray Brown, it should be noted that he was in fact Δ32 heterozygous prior to his HSCT, which likely provided him an advantage in relation to providing protection via Δ32 expression after transplantation.

The mechanisms of Berlin patient HIV cure are currently being investigated and pose an interesting question-is it a functional or sterilising cure? To start to answer this question, we will likely only be able to use the information currently available, as ongoing updates on 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 cure, it was derived from a functional cure approach.

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

, the median being 662/mm3

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

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

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].

, and cohort 2 was patients with

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

[75]. Patients

, the median being 272/mm3

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

autologous CD4+ T cells that were ZFN-modified. The

with CD4+ T cell count >450/mm3

comprise the virus reservoir.

**5.2. CRISPR/Cas9**

received one infusion containing 5x107

lower CD4+ T cells counts between 200 and 500/mm3

this off-target effect was determined to be deleterious.
