**2. HIV-1 life cycle: challenges for an HIV-1 cure**

HIV-1 can infect a wide range of cells, predominantly targeting CD4<sup>+</sup> T cells, dendritic cells, macrophages and other myeloid lineage cells [9]. This is achieved by binding of the viral envelope glycoprotein gp120 to the CD4 receptor, triggering a conformational change that allows for CCR5 or CXCR4 coreceptor binding. Further conformational changes in gp41 initiate a membrane fusion reaction that allows the viral capsid cytoplasmic entry [10]. Upon entry, the capsid disassembles to release viral RNA and proteins. To protect this genetic material, host restriction factors are subverted. With the aid of commandeered host cell machinery, a reverse transcription complex is formed and complementary viral DNA (cDNA) generated. The resulting pre-integration complex is transported via cytoskeletal manipulation to a nuclear pore complex where it is actively imported. The ability to traverse the nuclear membrane allows HIV to productively infect non-dividing cells. Viral integrase then facilitates the integration of viral DNA into the host genome to form the provirus [11]. Thus, HIV tethers its survival to the longevity of the cell and establishes the latent provirus reservoir, from which virus can reactivate. Using host replication machinery, viral RNA is then transcribed and exported to the cytoplasm. Proteins required for infectivity are synthesised and trafficked to the plasma membrane. Along with two RNA copies of the HIV genome, these proteins are assembled and packaged into immature virions in a process mediated by the viral Gag polyprotein. Once released from the plasma membrane, viral protease cleaves Gag into three structural proteins to create the mature infectious virion [12]. The HIV-1 life cycle is summarised in **Figure 1**.

While the above cycle is the most common model of HIV-1 infection, it is not exclusive. Numerous alternate pathways at varying stages of infection have been observed. For example, rather than the release of free HIV-1 virions, cell-to-cell transmission via infectious synapses can occur [13]. This modification is a more efficient means of infectivity [14]. Additionally, the above model does not account for infection of the central nervous system [15], in particular cells such as astrocytes, which lack the CD4 receptor [16]. While some of the proposed pathways remain controversial, HIV-1 is undeniably a versatile virus capable of hijacking diverse systems. The infectious route it takes may depend on the cell type, its available resources and activation status.

of anti-retroviral therapy (ART) and the more recent pre-exposure prophylaxis (PrEP), which has been embraced by many countries, particularly the US, UK and Australia [2], and demonstrated in landmark studies to prevent transmission by 99% [3, 4]. Additionally, the recent OPPOSITES ATTRACT study [5, 6] and PARTNER study [7] both showed that ART effectively reduces that rate of transmission to zero in homosexual male serodiscordant couples. However, while ART is highly effectively at controlling HIV-1 infection, it does not impact the latent reservoir, which is established early during virus infection. This allows the latent virus to recrudesce following any treatment interruption and results in rapid virus rebound to pretherapy levels. Therefore, ART does not constitute an HIV-1 cure. As is the case for many other diseases, gene therapy is being explored as an alternate HIV-1 treatment. There are, however, challenges specific to HIV, that may not arise in gene therapy approaches for other diseases. In the context of HIV, the latent reservoir represents a major barrier for developing an HIV cure, since ART has no effect on the integrated provirus and there is currently lack of a biomarker to identify cells that carry latent virus. Combined with the inability to identify latent cells and the

gene delivery platform uniquely challenging, especially in terms of a systemic *in vivo* approach. Another difficulty is that not all integrated provirus is the same, with some being full-length intact genomes and others being defective genomes that carry large deletions and will not result in productive infection following reactivation [8]. These are the barriers that must be addressed to ensure a gene therapy approach to HIV translates into patient outcomes. The unique chal-

macrophages and other myeloid lineage cells [9]. This is achieved by binding of the viral envelope glycoprotein gp120 to the CD4 receptor, triggering a conformational change that allows for CCR5 or CXCR4 coreceptor binding. Further conformational changes in gp41 initiate a membrane fusion reaction that allows the viral capsid cytoplasmic entry [10]. Upon entry, the capsid disassembles to release viral RNA and proteins. To protect this genetic material, host restriction factors are subverted. With the aid of commandeered host cell machinery, a reverse transcription complex is formed and complementary viral DNA (cDNA) generated. The resulting pre-integration complex is transported via cytoskeletal manipulation to a nuclear pore complex where it is actively imported. The ability to traverse the nuclear membrane allows HIV to productively infect non-dividing cells. Viral integrase then facilitates the integration of viral DNA into the host genome to form the provirus [11]. Thus, HIV tethers its survival to the longevity of the cell and establishes the latent provirus reservoir, from which virus can reactivate. Using host replication machinery, viral RNA is then transcribed and exported to the cytoplasm. Proteins required for infectivity are synthesised and trafficked to the plasma membrane. Along with two RNA copies of the HIV genome, these proteins are assembled and packaged into immature virions in a process mediated by the viral Gag polyprotein. Once released from the plasma membrane, viral protease cleaves Gag into three structural proteins to create the mature

lenges facing HIV-1 gene therapy and current solutions are described in this chapter.

cells) these hurdles make the development of a

T cells, dendritic cells,

rare frequency of latently-infected cells (~1 in 10<sup>6</sup>

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

**2. HIV-1 life cycle: challenges for an HIV-1 cure**

HIV-1 can infect a wide range of cells, predominantly targeting CD4<sup>+</sup>

infectious virion [12]. The HIV-1 life cycle is summarised in **Figure 1**.

**Figure 1.** HIV-1 life cycle and stages targeted by antiretroviral therapy (ART). ART drugs target various stages of the HIV life cycle, with some common drugs shown. Credit: National Institute of Allergy and Infectious Diseases (NIAID).

#### **2.1. The Life cycle and ART treatment**

Due to the inherent sequence variability of HIV-1 and the ability for virus resistance to arise, multiple stages in the virus life cycle need to be targeted by ART to control the infection. As illustrated in **Figure 1**, stages that are targeted can include binding, fusion, reverse transcriptions and integration, among others, with either one or more ART drugs from each stage being utilised to provide sufficient HIV-1 control. An example of one such drug combination is TRUVADA®, which combines two drugs targeting the reverse transcription stage, emtricitabine and tenofovir and has been widely embraced for PrEP treatment.

**3.1. Shock and kill**

process is shown in **Figure 2**.

*3.2.1. Tat-inhibitor didehydro-cortistatin A (dCA)*

HIV cure, and to increase the longevity of the suppressive effect.

**3.2. Block and lock**

**Figure 2**.

The most studied sterilising approach is aptly named "shock and kill". This concept explores the use of *latency reversing agents* (LRAs) to "shock" the virus into reactivation, whereby it is detected and "killed" by either its own cytotoxicity or the host immune system. To date, this purging strategy has been largely unsuccessful. Proposed agents either induce global immune reactivation, leading to increased pro-inflammatory cytokines and severe side effects, or *in vitro* efficacy has simply failed to translate *in vivo* [27–32]. Extensive descriptions of LRAs and the next step of optimising the "kill" step is reviewed by Kim et al., [33] and are not the focus of this chapter. However, one recent study has demonstrated a specific kill response mediated by ricin A, which is initially encapsulated in a novel nanocapsule polymer shell and then activated and released via the ricin A peptide crosslinkers being cleaved by HIV-1 protease [34]. Some of the LRAs under investigation are summarised in **Table 1** and a schematic of the

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

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

The limited success of sterilising cures has shifted the priority to identifying a functional cure; which is now seen as a more realistic approach to controlling the viral reservoir, without ART. The functional cure approach is termed "block and lock". This concept exploits the use of *latency inducing agents* (LIAs) to "block" virus gene transcription at the promoter via epigenetic mechanisms and "lock" the integrated virus genome in a permanent super-latent state, which resembles the natural latent reservoir. We and others are pursuing this approach to provide a sustained virus remission, without ART. Some of the LIAs under investigation are summarised in **Table 1** and a schematic of the process is shown in

In 2012, the Valente group reported a novel small molecule inhibitor of HIV transcription, termed Didehydro-cortistatin A (dCA) [35]. Derived from a marine sponge, dCA inhibits Tat-mediated transactivation of integrated HIV provirus via disrupting binding of the transactivation response (TAR) element to Tat through direct binding competition with the RNA hairpin TAR-binding domain of Tat. Effectively disrupting the Tat/TAR complex, dCA induced prolonged suppression of virus transcription in HeLa-CD4 cells infected with HIV-1NL4.3 for 2 months with constant treatment and out to a further 27 days following cessation of treatment, as determined by measuring viral RNA levels. The inhibitory effect of dCA on HIV replication *ex vivo* in primary CD4+ T cells isolated from eight HIV-1 infected individuals on suppressive ART was ~60% compared to no treatment, while ART treatment alone reduced viral production to ~40% and dCA treatment alone reduced virus production to ~25% [35]. The study reported there were no apparent cytotoxic effects in cell culture models or when assessed in C57BI-6 mice at the concentrations used. Further optimisation of dCA will be required if the treatment is to be effective in the absence of ART, as desired for a functional

#### **2.2. The latent reservoir**

Although the versatility of HIV-1 presents a challenge for the development of therapeutics, by far, the latent cellular reservoirs are the greatest barrier to developing a cure. The difficulties in controlling these virus reservoirs arise when the infection becomes latent. While the exact mechanisms of HIV latency are still being precisely defined, studies have demonstrated epigenetic regulation is involved in suppressing virus transcription, with the presence of classic epigenetic repressive marks, including methylation (*i.e.* histone 3 lysine 27 trimethylation (H3K27me3)) and deacetylation (*i.e.* Histone deacetylase 1 (HDAC1)) on N-terminal histone tails inducing specific epigenetic chromatin compaction, termed heterochromatin [17].

The latent reservoir resides in resting memory CD4+ T cells [18, 19], such as central memory, effector memory and transitional memory cells [8], T follicular helper cells [20], macrophages and other myeloid cells, as well as in immune privileged sanctuary sites, such as the gut [21], lymph nodes and associated germinal centers and the brain. Physiologically, in their resting states CD4+ T cells have low endocytic and metabolic rates that are sufficient for maintenance of housekeeping functions [22]. As such, they are not impacted by ART. To retain dormancy, they negatively regulate gene activation via inhibition of cellular transcription factors, such as NF-κB and NFAT [23]: host factors essential for initiating active HIV-1 virus production [24–26]. Consequently, the integrated provirus is reversibly silenced by epigenetic repression and evades host immune detection. This presents a clinically daunting prospect. As memory cells are long lived, with ART controlling the infection, not only would eradication of the reservoir take over 70 years [18], but theoretically, one infected cell could sufficiently sustain a life-long infection. Further, upon reactivation of the infected cell/s by any stimuli, virus recrudescence rapidly occurs and thus while ART can effectively control HIV-1 infection, it does not represent a cure.

#### **3. HIV-1 cure strategies**

Various approaches to overcome the viral reservoir barrier are being pursued. Primarily, they can be separated into two main categories: sterilising and functional. Both approaches aim for an undetectable viral load without the need for ART, with the sterilising approach being defined as complete eradication of the virus, and conversely, the functional approach is defined as controlling the virus reservoir without its eradication.

#### **3.1. Shock and kill**

**2.1. The Life cycle and ART treatment**

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

**2.2. The latent reservoir**

does not represent a cure.

**3. HIV-1 cure strategies**

Due to the inherent sequence variability of HIV-1 and the ability for virus resistance to arise, multiple stages in the virus life cycle need to be targeted by ART to control the infection. As illustrated in **Figure 1**, stages that are targeted can include binding, fusion, reverse transcriptions and integration, among others, with either one or more ART drugs from each stage being utilised to provide sufficient HIV-1 control. An example of one such drug combination is TRUVADA®, which combines two drugs targeting the reverse transcription stage, emtric-

Although the versatility of HIV-1 presents a challenge for the development of therapeutics, by far, the latent cellular reservoirs are the greatest barrier to developing a cure. The difficulties in controlling these virus reservoirs arise when the infection becomes latent. While the exact mechanisms of HIV latency are still being precisely defined, studies have demonstrated epigenetic regulation is involved in suppressing virus transcription, with the presence of classic epigenetic repressive marks, including methylation (*i.e.* histone 3 lysine 27 trimethylation (H3K27me3)) and deacetylation (*i.e.* Histone deacetylase 1 (HDAC1)) on N-terminal histone tails inducing specific epigenetic chromatin compaction, termed heterochromatin [17].

The latent reservoir resides in resting memory CD4+ T cells [18, 19], such as central memory, effector memory and transitional memory cells [8], T follicular helper cells [20], macrophages and other myeloid cells, as well as in immune privileged sanctuary sites, such as the gut [21], lymph nodes and associated germinal centers and the brain. Physiologically, in their resting states CD4+ T cells have low endocytic and metabolic rates that are sufficient for maintenance of housekeeping functions [22]. As such, they are not impacted by ART. To retain dormancy, they negatively regulate gene activation via inhibition of cellular transcription factors, such as NF-κB and NFAT [23]: host factors essential for initiating active HIV-1 virus production [24–26]. Consequently, the integrated provirus is reversibly silenced by epigenetic repression and evades host immune detection. This presents a clinically daunting prospect. As memory cells are long lived, with ART controlling the infection, not only would eradication of the reservoir take over 70 years [18], but theoretically, one infected cell could sufficiently sustain a life-long infection. Further, upon reactivation of the infected cell/s by any stimuli, virus recrudescence rapidly occurs and thus while ART can effectively control HIV-1 infection, it

Various approaches to overcome the viral reservoir barrier are being pursued. Primarily, they can be separated into two main categories: sterilising and functional. Both approaches aim for an undetectable viral load without the need for ART, with the sterilising approach being defined as complete eradication of the virus, and conversely, the functional approach is

defined as controlling the virus reservoir without its eradication.

itabine and tenofovir and has been widely embraced for PrEP treatment.

The most studied sterilising approach is aptly named "shock and kill". This concept explores the use of *latency reversing agents* (LRAs) to "shock" the virus into reactivation, whereby it is detected and "killed" by either its own cytotoxicity or the host immune system. To date, this purging strategy has been largely unsuccessful. Proposed agents either induce global immune reactivation, leading to increased pro-inflammatory cytokines and severe side effects, or *in vitro* efficacy has simply failed to translate *in vivo* [27–32]. Extensive descriptions of LRAs and the next step of optimising the "kill" step is reviewed by Kim et al., [33] and are not the focus of this chapter. However, one recent study has demonstrated a specific kill response mediated by ricin A, which is initially encapsulated in a novel nanocapsule polymer shell and then activated and released via the ricin A peptide crosslinkers being cleaved by HIV-1 protease [34]. Some of the LRAs under investigation are summarised in **Table 1** and a schematic of the process is shown in **Figure 2**.

#### **3.2. Block and lock**

The limited success of sterilising cures has shifted the priority to identifying a functional cure; which is now seen as a more realistic approach to controlling the viral reservoir, without ART. The functional cure approach is termed "block and lock". This concept exploits the use of *latency inducing agents* (LIAs) to "block" virus gene transcription at the promoter via epigenetic mechanisms and "lock" the integrated virus genome in a permanent super-latent state, which resembles the natural latent reservoir. We and others are pursuing this approach to provide a sustained virus remission, without ART. Some of the LIAs under investigation are summarised in **Table 1** and a schematic of the process is shown in **Figure 2**.

#### *3.2.1. Tat-inhibitor didehydro-cortistatin A (dCA)*

In 2012, the Valente group reported a novel small molecule inhibitor of HIV transcription, termed Didehydro-cortistatin A (dCA) [35]. Derived from a marine sponge, dCA inhibits Tat-mediated transactivation of integrated HIV provirus via disrupting binding of the transactivation response (TAR) element to Tat through direct binding competition with the RNA hairpin TAR-binding domain of Tat. Effectively disrupting the Tat/TAR complex, dCA induced prolonged suppression of virus transcription in HeLa-CD4 cells infected with HIV-1NL4.3 for 2 months with constant treatment and out to a further 27 days following cessation of treatment, as determined by measuring viral RNA levels. The inhibitory effect of dCA on HIV replication *ex vivo* in primary CD4+ T cells isolated from eight HIV-1 infected individuals on suppressive ART was ~60% compared to no treatment, while ART treatment alone reduced viral production to ~40% and dCA treatment alone reduced virus production to ~25% [35]. The study reported there were no apparent cytotoxic effects in cell culture models or when assessed in C57BI-6 mice at the concentrations used. Further optimisation of dCA will be required if the treatment is to be effective in the absence of ART, as desired for a functional HIV cure, and to increase the longevity of the suppressive effect.



P-TEFb, positive transcription elongation factor b; TLR, Toll-like receptor; mTOR, mechanistic target of rapamycin; STAT5, signal transducer and activator of transcription 5; IL-15, interleukin-15; dCA, Didehydro-cortistatin A; UHRF1, Ubiquitin-like, containing PHD and RING finger domains 1 protein.

PTGS was the first distinct pathway to be discovered in 1998 by Fire and Mello [39]. It can be considered the predominantly cytoplasmic arm of RNAi. Briefly, short interfering RNAs (siRNAs) 19–23 base pairs long are processed by the Dicer endonuclease and loaded onto the Argonaute 2 (Ago2) protein. Several of other proteins are recruited to form the RNA-induced silencing complex (RISC). This complex uses the siRNA as complementary anti-sense guides to identify target mRNA in the cytoplasm that are subsequently cleaved and degraded by the catalytic activity of Ago2 [41]. As a result, target gene expression is transiently downregulated prior to translation. Exhibiting enormous potential, PTGS has since been developed as a tool to selectively inhibit the expression of critical HIV-1 viral proteins *in vitro,* leading to significant reductions in viral replication [42–46]. The sequence specific nature of the PTGS process suggests there are minimal off-target effects [47], although these can still occur. Some reports have shown that introducing exogenous siRNA less than 24 base pairs in length does not trigger an interferon mediated immune response or defence mechanism to the synthetic material [48].

**Figure 2.** "Shock and kill" versus "Block and Lock" HIV cure strategies. Latency reversing agents (LRAs) activate or "shock" the HIV-1 provirus awake with the aim that the infected cell will then undergo "kill" via cytotoxic or host immune mechanisms. Latency inducing agents (LIAs) mediate the opposite effect, by "blocking" virus replication and "locking" the virus genome in an induced "super-latency" that is refractory to reactivation. HDAC, histone deacetylase; HMT, histone methyl transferase; PKC, protein kinase C; dCA, didehydro-cortistatin A; ART, antiretroviral therapy.

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

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

While these are all highly desirable traits for future gene therapeutics, PTGS has several limitations, particularly in the context of HIV-1, which leave it unsuitable for gene therapy. A consequence of its high specificity is that a nucleotide mismatches between the siRNA and its target could be sufficient to abrogate silencing [49]. Hence, in the context of a highly diverse virus with multiple subtypes, a conserved target sequence must be carefully selected. Due

**Table 1.** Agents that modulate HIV latency.

#### *3.2.2. RNA silencing*

RNA interference (RNAi) is a fundamentally conserved process crucial for viral defence and the regulation of normal gene expression. Since its initial discovery in transgenic tobacco plants [36], the RNAi field has erupted with literature exploring the depth of its possibilities. From a tool to study basic gene functions, to a remedy for previously untreatable conditions, RNAi has the potential to revolutionise research and medicine. As a result, it has been extensively studied and characterised in a wide array of organisms, particularly plants (*Arabidopsis thaliana* [37, 38]), the nematode *Caenorhabditis elegans* [39] and fission yeast *Schizosaccharomyces pombe* (*S*. *pombe*) [40]. Two distinct pathways have since emerged: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS); also termed epigenetic silencing (refer to **Figure 3** for an overview).

**Figure 2.** "Shock and kill" versus "Block and Lock" HIV cure strategies. Latency reversing agents (LRAs) activate or "shock" the HIV-1 provirus awake with the aim that the infected cell will then undergo "kill" via cytotoxic or host immune mechanisms. Latency inducing agents (LIAs) mediate the opposite effect, by "blocking" virus replication and "locking" the virus genome in an induced "super-latency" that is refractory to reactivation. HDAC, histone deacetylase; HMT, histone methyl transferase; PKC, protein kinase C; dCA, didehydro-cortistatin A; ART, antiretroviral therapy.

PTGS was the first distinct pathway to be discovered in 1998 by Fire and Mello [39]. It can be considered the predominantly cytoplasmic arm of RNAi. Briefly, short interfering RNAs (siRNAs) 19–23 base pairs long are processed by the Dicer endonuclease and loaded onto the Argonaute 2 (Ago2) protein. Several of other proteins are recruited to form the RNA-induced silencing complex (RISC). This complex uses the siRNA as complementary anti-sense guides to identify target mRNA in the cytoplasm that are subsequently cleaved and degraded by the catalytic activity of Ago2 [41]. As a result, target gene expression is transiently downregulated prior to translation. Exhibiting enormous potential, PTGS has since been developed as a tool to selectively inhibit the expression of critical HIV-1 viral proteins *in vitro,* leading to significant reductions in viral replication [42–46]. The sequence specific nature of the PTGS process suggests there are minimal off-target effects [47], although these can still occur. Some reports have shown that introducing exogenous siRNA less than 24 base pairs in length does not trigger an interferon mediated immune response or defence mechanism to the synthetic material [48].

*3.2.2. RNA silencing*

**Latency reversing agents (LRAs)**

**Class Agent**

*Protein Kinase C Agonists* Prostratin/Bryostatin

*TCR Activators* Immune checkpoint blockers

*Epigenetic Modifiers* Didehydro-cortistatin A (dCA)

Ubiquitin-like, containing PHD and RING finger domains 1 protein.

TLR9

siRNA shRNA

Nullbasic

*PI3K/Akt pathway* Disulfiram

*Cytokines* IL-15 *TLR Agonists* TLR7

**Class Agent**

*HIV Integrase inhibitors* LEDGINs

**Table 1.** Agents that modulate HIV latency.

**Latency inducing agents (LIAs)**

*Epigenetic Modifiers* Histone deacetylase inhibitors (HDACs)

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

Methylation inhibitors Methyl-transferase inhibitors Bromodomain inhibitors P-TEFb activators

Ingenol B/PEP0005

STATS signalling benzotriazole mTOR complex Rapamycin

(refer to **Figure 3** for an overview).

RNA interference (RNAi) is a fundamentally conserved process crucial for viral defence and the regulation of normal gene expression. Since its initial discovery in transgenic tobacco plants [36], the RNAi field has erupted with literature exploring the depth of its possibilities. From a tool to study basic gene functions, to a remedy for previously untreatable conditions, RNAi has the potential to revolutionise research and medicine. As a result, it has been extensively studied and characterised in a wide array of organisms, particularly plants (*Arabidopsis thaliana* [37, 38]), the nematode *Caenorhabditis elegans* [39] and fission yeast *Schizosaccharomyces pombe* (*S*. *pombe*) [40]. Two distinct pathways have since emerged: post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS); also termed epigenetic silencing

P-TEFb, positive transcription elongation factor b; TLR, Toll-like receptor; mTOR, mechanistic target of rapamycin; STAT5, signal transducer and activator of transcription 5; IL-15, interleukin-15; dCA, Didehydro-cortistatin A; UHRF1,

Ubiquitin-like, containing PHD and RING finger domains 1 protein (UHRF1)

While these are all highly desirable traits for future gene therapeutics, PTGS has several limitations, particularly in the context of HIV-1, which leave it unsuitable for gene therapy. A consequence of its high specificity is that a nucleotide mismatches between the siRNA and its target could be sufficient to abrogate silencing [49]. Hence, in the context of a highly diverse virus with multiple subtypes, a conserved target sequence must be carefully selected. Due

circumvent a single PTGS therapeutic. Similar to ART, current PTGS therapeutics are having to be combined, to simultaneously target multiple HIV proteins and/or to target host targets,

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

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

In comparison, TGS can be considered the nucleic arm of RNAi. It offers the highly specific targeting of the integrated HIV provirus. Still controversial in mammals, this pathway begins in the cytoplasm where siRNA associate with Argonaute 1 protein (Ago1). These two components are trafficked to the nucleus [53] and recruit other proteins to form the RNA-induced transcriptional silencing (RITS) complex. The RITS protein components have been identified in *S. pombe* yeast and include Ago1, the GW protein, Tas3, and chromodomain protein 1 (Chp1) [54], however while Ago1 is present in mammalian cells, Tas3 and Chp1 homologues have not yet been identified. Although this complex can be considered as the equivalent of RISC from PTGS, it is not identical, due to the different functional requirements and distinct protein components. Via siRNA sequence complementarity, RITS identifies the target locus and induces chromatin compaction through epigenetic modifications, such as histone methylation [55]. By rendering it structurally inaccessible to transcriptional machinery, TGS can lock the virus in a latent state. Like PTGS, TGS is capable of significantly suppressing HIV-1 production and is highly sequence specific, with minimal off-target effects and interferon mediated immune responses or defence mechanisms, dependent on the specific sequence targeted [56]. A single nucleotide mismatch between the siRNA and its target could be sufficient to disrupt silencing. Hence, conserved regions of the provirus must be carefully selected and a multiplexed approach may be necessary. TGS also offers several advantages over its cytoplasmic counterpart. By preventing the provirus from actively transcribing, it can silence HIV-1 prior to the generation of escape variants. Additionally, due to the heritable nature of the heterochromatin marks,

Our laboratory has described two siRNA sequences capable of inducing potent TGS in HIVinfected cells. The first siRNA, termed siPromA, was identified in 2005 and has been extensively characterised as inducing highly-sequence specific TGS via epigenetic repressive mechanisms [53, 55, 56, 58–60]. The siPromA sequence targets NF-κB tandem repeat motif, which is unique to the virus and is not homologous to any host cell NF-κB motifs. This is important as NF-κB is an important transcription factor for multiple cell signalling pathways. A second siRNA, termed si143, has recently been shown to also induce TGS and targets the COUP-TF and AP-1 transcription factor sites upstream of the siPromA target sequence [61]. When combined, these two siRNAs provide enhanced suppression and enforcement of latency through multiple epigenetic modifications. We have shown up to 1000-fold suppression of virus transcription following a single transfection of siPromA or si143 for up to 15 days in various HIV-infected cell cultures, including HeLa T4+ cells and Hut78 cells. Further, in MOLT-4 cells carrying shRNA expressing siPromA, we reported virus transcription was suppressed for over 1 year [60]. Specific epigenetic modifications in HIV cultures suppressed by siPromA or si143 have been investigated using ChIP assays and included enrichment of histone methylation on the N-terminal histone tail (such as HeK27me3, H3K9me2) and decreased acetylation (such as H3K9) [61]. We have also demonstrated effective *in vivo* virus suppression using a humanised mouse model of acute HIV in (NOD)/SCID/Janus kinase 3 (NOJ) knockout mice infected with

such as CCR5, to overcome the generation of resistance mutations [52].

daughter cells exhibit the same suppressive phenotype [57].

*3.2.2.1. RNA-directed latency inducing agents*

**Figure 3.** Gene silencing pathways mediated by siRNA. siRNA duplexes induce posttranscriptional gene silencing (PTGS) predominately in the cytoplasm through RISC machinery initiating mRNA degradation, and transcriptional gene silencing (TGS) in the nucleus through the RITS complex inducing chromatin compaction to silence gene expression. Ago1, Argonaute 1; Ago2, Argonaute 2; shRNA, short hairpin RNA; RISC, RNA induced silencing complex; RITS, RNAinduced transcriptional silencing complex; TRBP, trans-activating response RNA-binding protein; TNRC6, trinucleotide repeat containing six proteins.

to the enormous diversity of the genome, a multiplexed approach would be necessary to provide adequate coverage across HIV subtypes and strains. Additionally, as its silencing effects are only transient, PTGS would require either (1) frequent siRNA administration to an infected individual, thus rendering it a recurring treatment and not a cure, or (2) provision of a self-sustaining system. This would involve viral vector delivery of a plasmid that can constitutively express target siRNAs. This may be in the form of short hairpin (sh)RNAs that are exported to the cytoplasm and processed into the desired siRNA. The greatest pitfall however, is that PTGS predominately functions in the cytoplasm, targeting mRNA from an actively transcribing provirus. As such, it cannot overcome the selective pressures driving HIV-1 mutation. Rather, this process allows the virus to rapidly transcribe escape variants [50]. From point mutations and deletions that disrupt target specificity, to the generation of alternative secondary structures to prevent RISC accessibility [51], HIV-1 can rapidly circumvent a single PTGS therapeutic. Similar to ART, current PTGS therapeutics are having to be combined, to simultaneously target multiple HIV proteins and/or to target host targets, such as CCR5, to overcome the generation of resistance mutations [52].

#### *3.2.2.1. RNA-directed latency inducing agents*

to the enormous diversity of the genome, a multiplexed approach would be necessary to provide adequate coverage across HIV subtypes and strains. Additionally, as its silencing effects are only transient, PTGS would require either (1) frequent siRNA administration to an infected individual, thus rendering it a recurring treatment and not a cure, or (2) provision of a self-sustaining system. This would involve viral vector delivery of a plasmid that can constitutively express target siRNAs. This may be in the form of short hairpin (sh)RNAs that are exported to the cytoplasm and processed into the desired siRNA. The greatest pitfall however, is that PTGS predominately functions in the cytoplasm, targeting mRNA from an actively transcribing provirus. As such, it cannot overcome the selective pressures driving HIV-1 mutation. Rather, this process allows the virus to rapidly transcribe escape variants [50]. From point mutations and deletions that disrupt target specificity, to the generation of alternative secondary structures to prevent RISC accessibility [51], HIV-1 can rapidly

repeat containing six proteins.

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

**Figure 3.** Gene silencing pathways mediated by siRNA. siRNA duplexes induce posttranscriptional gene silencing (PTGS) predominately in the cytoplasm through RISC machinery initiating mRNA degradation, and transcriptional gene silencing (TGS) in the nucleus through the RITS complex inducing chromatin compaction to silence gene expression. Ago1, Argonaute 1; Ago2, Argonaute 2; shRNA, short hairpin RNA; RISC, RNA induced silencing complex; RITS, RNAinduced transcriptional silencing complex; TRBP, trans-activating response RNA-binding protein; TNRC6, trinucleotide In comparison, TGS can be considered the nucleic arm of RNAi. It offers the highly specific targeting of the integrated HIV provirus. Still controversial in mammals, this pathway begins in the cytoplasm where siRNA associate with Argonaute 1 protein (Ago1). These two components are trafficked to the nucleus [53] and recruit other proteins to form the RNA-induced transcriptional silencing (RITS) complex. The RITS protein components have been identified in *S. pombe* yeast and include Ago1, the GW protein, Tas3, and chromodomain protein 1 (Chp1) [54], however while Ago1 is present in mammalian cells, Tas3 and Chp1 homologues have not yet been identified. Although this complex can be considered as the equivalent of RISC from PTGS, it is not identical, due to the different functional requirements and distinct protein components. Via siRNA sequence complementarity, RITS identifies the target locus and induces chromatin compaction through epigenetic modifications, such as histone methylation [55]. By rendering it structurally inaccessible to transcriptional machinery, TGS can lock the virus in a latent state.

Like PTGS, TGS is capable of significantly suppressing HIV-1 production and is highly sequence specific, with minimal off-target effects and interferon mediated immune responses or defence mechanisms, dependent on the specific sequence targeted [56]. A single nucleotide mismatch between the siRNA and its target could be sufficient to disrupt silencing. Hence, conserved regions of the provirus must be carefully selected and a multiplexed approach may be necessary. TGS also offers several advantages over its cytoplasmic counterpart. By preventing the provirus from actively transcribing, it can silence HIV-1 prior to the generation of escape variants. Additionally, due to the heritable nature of the heterochromatin marks, daughter cells exhibit the same suppressive phenotype [57].

Our laboratory has described two siRNA sequences capable of inducing potent TGS in HIVinfected cells. The first siRNA, termed siPromA, was identified in 2005 and has been extensively characterised as inducing highly-sequence specific TGS via epigenetic repressive mechanisms [53, 55, 56, 58–60]. The siPromA sequence targets NF-κB tandem repeat motif, which is unique to the virus and is not homologous to any host cell NF-κB motifs. This is important as NF-κB is an important transcription factor for multiple cell signalling pathways. A second siRNA, termed si143, has recently been shown to also induce TGS and targets the COUP-TF and AP-1 transcription factor sites upstream of the siPromA target sequence [61]. When combined, these two siRNAs provide enhanced suppression and enforcement of latency through multiple epigenetic modifications. We have shown up to 1000-fold suppression of virus transcription following a single transfection of siPromA or si143 for up to 15 days in various HIV-infected cell cultures, including HeLa T4+ cells and Hut78 cells. Further, in MOLT-4 cells carrying shRNA expressing siPromA, we reported virus transcription was suppressed for over 1 year [60]. Specific epigenetic modifications in HIV cultures suppressed by siPromA or si143 have been investigated using ChIP assays and included enrichment of histone methylation on the N-terminal histone tail (such as HeK27me3, H3K9me2) and decreased acetylation (such as H3K9) [61]. We have also demonstrated effective *in vivo* virus suppression using a humanised mouse model of acute HIV in (NOD)/SCID/Janus kinase 3 (NOJ) knockout mice infected with 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.

[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 homo-

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

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

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

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

memory, was almost paralysed and had to learn to walk again [65, 71].

zygous CCR5-delta32 allele was identified [66].

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 people living with HIV globally.

#### *3.2.2.2. RNA-aptamer silencing*

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].
