**6. Delivery**

#### **6.1.** *Ex vivo* **delivery by lentiviral vectors**

Delivery of gene therapy to a specific target cell is another current challenge for an HIV cure. Viral vectors have become a regular method by which to deliver therapeutic genes and constructs [85]. There are multiple viable types of viral vectors that have been proven to be safe, relatively easy to construct and modify, and in the case of lentiviral vectors, these have the potential to transduce cells in a non-proliferative state [86]. Although the latter feature does not extend to non-proliferative leukocytes, due the presence of lentiviral restriction factors at and below the membrane.

One significant obstacle to the effective delivery of sufficient quantities viral vector is the ability to transduce sufficient quantities of target cells. To overcome this, apheresis is performed in order to concentrate the desired cells. Currently, gene therapy protocols for HIV first require the isolation of the desired cells to be modified, typically following apheresis [85]. Apheresis is the process of removing mononuclear cells from blood and returning neutrophils, platelets, plasma and red blood cells to the donor. This process is performed in order to collect more of the desired cells of the blood than could be separated from a unit of whole blood of ~550 mL. While CD4+ T cells are the main target for HIV infection, other cells such as dendritic cells, macrophages, monocytes and to a lesser extent, haematopoietic stem cells (HSC) have been found to be susceptible to HIV infection [87–89]. It is known that if HSC are transduced, or modified in any way, then a wide range of subsequent immune cells including macrophages, dendritic cells, CD4+ T cells and NK cells will carry that modification [90]. While transduction of CD4+ T cells will result in only CD4+ T cells being modified, the approach of transducing HSC provides protection from HIV to a broader range of cell-types, making it a highly desirable target for treatment/modification. Once a large volume of cells has been collected over several hours, they can then be transduced with the desired viral vector and reintroduced to the individual where the cells will migrate back to peripheral blood, lymph nodes, and bone marrow. This delivery method has been used in dozens of clinical trials and has become a widely accepted method for delivering viral vectors to large numbers of cells, in particular to HSC in the bone marrow [85]. The *ex vivo* gene therapy process is depicted in **Figure 4**.

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

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"

Delivery of gene therapy to a specific target cell is another current challenge for an HIV cure. Viral vectors have become a regular method by which to deliver therapeutic genes and constructs [85]. There are multiple viable types of viral vectors that have been proven to be safe, relatively easy to construct and modify, and in the case of lentiviral vectors, these have the potential to transduce cells in a non-proliferative state [86]. Although the latter feature does not extend to non-proliferative leukocytes, due the presence of lentiviral restriction factors at

One significant obstacle to the effective delivery of sufficient quantities viral vector is the ability to transduce sufficient quantities of target cells. To overcome this, apheresis is performed in order to concentrate the desired cells. Currently, gene therapy protocols for HIV first require the isolation of the desired cells to be modified, typically following apheresis [85]. Apheresis is the process of removing mononuclear cells from blood and returning neutrophils, platelets, plasma and red blood cells to the donor. This process is performed in order to collect more of the desired cells of the blood than could be separated from a unit of whole blood of ~550 mL. While CD4+ T cells are the main target for HIV infection, other cells such as dendritic cells, macrophages, monocytes and to a lesser extent, haematopoietic stem cells (HSC) have been found to be susceptible to HIV infection [87–89]. It is known that if HSC are transduced, or modified in any way, then a wide range of subsequent immune cells including macrophages, dendritic cells, CD4+ T cells and NK cells will carry that modification [90]. While transduction of CD4+ T cells will result in only CD4+ T cells being modified, the approach of transducing HSC provides protection from HIV to a broader range of cell-types, making it a highly desirable target for treatment/modification. Once a large volume of cells has been collected over several hours, they can then be transduced with the desired viral vector and reintroduced to the individual where the cells will migrate back to peripheral blood, lymph nodes, and bone marrow. This delivery method has been used in dozens of clinical trials and has become a

latency marker.

**6. Delivery**

and below the membrane.

approach, targets the NF-κB binding motif.

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

**6.1.** *Ex vivo* **delivery by lentiviral vectors**

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.

be expected due to the required production of cells, thus delaying the effect of the therapeutic gene(s). Production of new CD4+ T lymphocytes from the thymus has been predicted to be at a rate of approximately 1.65 cells/μL of blood/day due to thymic function [95]. The resulting in the production of a stable population of protected cells would be expected to gradually create a positive impact on CD4+ T cell number and help suppress viral load.

To overcome this lack of secondary lymphoid targeting of CAR CD8+ T cells, the Skinner laboratory has recently developed a hybrid CAR construct that encompasses not only HIV targeting, but also the CXCR5 receptor [101]. In theory, this enables CD8+ T cells not only to

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

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

One vector of note that has been extensively studied is the Cal-1 vector, which uses both the maC46 fusion inhibitor and shRNA-CCR5. This construct has been extensively studied, consistently showing therapeutic benefits *in vitro*. Additionally, this construct has also shown its enhanced efficacy when compared against individual genes, as the effect is induced by the use of two therapeutic targets [102]. This has not only led to stronger protection from HIV infection, but also is likely to result in reduced risk of mutation resistance [102]. This has been examined in mouse studies and non-human primates, where it has shown safety, high levels of engraftment (including in CD34+ cells), and a selective growth advantage [102–105].

The Cal-1 therapeutic construct is currently undergoing Phase I/II clinical trials [106]. The study involves 12 HIV positive patients, which have undergone transduction of both HSC and CD4+ T cells with a lentiviral vector carrying both the shRNA-CCR5 and C46 fusion inhibitor. The patients were divided into 3 equal groups, group 1 received no busulfan preconditioning, group 2 received 4 mg/kg busulfan, and group 3 received 2 doses to a total of 6 mg/kg busulfan conditioning. This study is currently ongoing but will provide important

Whilst we now have therapeutic approaches that can focus our efforts on a HIV cure, delivery of these components still presents a barrier. Lentiviral vectors have proven to be extremely useful in providing delivery of therapeutic genes, although there are still limitations. As mentioned, cells can only be modified *ex vivo*, thus requiring apheresis. Additionally, in the case of HIV, as ART will prevent uptake of the lentiviral vector, patients must first stop ART, thus raising various health concerns and ethical obstacles. Furthermore, current approved lentiviral platforms can only transduce T cells that are activated, as this over comes lentiviral restrictions at the membrane and underneath the membrane. The sum of these problems significantly increases the cost of the clinical approach. In the setting of CAR-T cells the estimates for treatment of refractory B cell leukaemia is approximately \$US400,000. Given this substantial cost, the accessibility of this type of therapeutic intervention is low. Thus, efforts are underway that will improve the process of this gene delivery pipeline. For instance, lentiviral vectors could be developed to target fresh leukocyte populations *ex vivo*, obviating the need for large scale apheresis. Additionally, lentiviral vectors could be modified to target leukocyte subsets, so the cells with the greatest stem-like attributes are re-infused and not diluted with cells that may not proceed down the differentiation pathway. This could potentially include a sub-population of resting T cells (*e.g.* Stem T cells) being isolated, genetically modified and re-infused in a manner that may not require apheresis. However, whether re-infusion of a smaller population of stem T cell would result in the same outcome that maybe achieved with a large population

data on the optimised conditioning treatment to guide future treatment studies.

of bulk T cells obtained by leukopheresis needs to be thoroughly investigated.

target the HIV reservoir, but also transverse the site where the reservoir is located.

*6.1.1. Cal-1 lentiviral vector*

*6.1.2. Limitations of lentiviral vector delivery*

While the modification of HSC has the benefit of long-lasting and broad-spectrum protection via the differentiation of stem cells, this approach still lacks the immediate benefit of targeting the existing CD4+ T cells population. The use of CD4+ T cells as a target for HIV gene therapy has been explored and assessed in several studies. Isolation and modification of CD4+ T cells is relatively simple, as they largely populate and consistently traffic through peripheral blood. Accordingly, no stimulatory factors (such as G-CSF) are required to mobilise them prior to collection. This method has the benefits of providing an immediate benefit via the reintroduction of a protected population of the primary target cells for HIV infection [96]. This has been performed and shown to be both safe in treatment, and effective in delivery of the therapeutic gene [96, 97].

Lentiviral vectors are being increasingly used in clinical trials to treat a variety of diseases ranging from cancers, to genetic diseases such as haemophilia and sickle cell anaemia, as well as several trials treating HIV. The largest such trial in HIV gene therapy demonstrating the safety of lentiviral vectors was the Phase II trial whereby a Tat/Vpr specific anti-HIV ribozyme (OZ1) or placebo was delivered in autologous CD34+ haematopoietic progenitor cells. The trial involved 74 patients where there were no adverse events related to the vectors or infusion process [98].

As outlined above, present gene therapy efforts to target HIV are primarily defensive in approach, as they encode future HIV resistance and may not influence the HIV reservoir in the short-term. Given the success of CAR T cell therapy in various cancer trials, many investigators are now multiplexing HIV resistance alongside a CAR construct that can target HIV. As cellular markers for the HIV reservoir are often shared in various leukocyte niches, the equivalent to the anti-CD19 approach used in B cell leukaemia has yet to be determined. Rather, investigators have now turned to potent broadly neutralising antibodies, which have been screened and cloned from various HIV positive patients and target HIV envelope. In this setting, several pre-clinical studies are underway in non-human primates in the laboratories of Kiem and Jerome, where resistance afforded by C46 and shRNA is complexed with one of several CAR modules that incorporates the single variable change of well characterised broadly neutralising antibodies [99].

In contrast, work led by the Berger laboratory has taken a similar but different approach to CAR T cell development. Rather than incorporating a neutralising antibody, they have complexed the first Ig-Like domains of CD4 with the serum mannose binding lectin [100]. This approach enables global recognition of HIV envelope, as it engages the CD4 binding site and also the abundant glycosylation sites that decorate the antigenic silent face of HIV Env. In the CAR T cell context, CD8+ T cells are generated alongside HIV resistant CD4+ T cells to mediate attack on the HIV reservoir. The only problem with the latter approach is that the major HIV reservoir *in vivo* resides in the germinal centers of secondary lymphoid tissue and actively excludes CD8+ T cells, given they lack the germinal homing receptor CXCR5. Therefore, whilst CD4+ T cells may transverse the germinal center, CD8+ T cells will not. To overcome this lack of secondary lymphoid targeting of CAR CD8+ T cells, the Skinner laboratory has recently developed a hybrid CAR construct that encompasses not only HIV targeting, but also the CXCR5 receptor [101]. In theory, this enables CD8+ T cells not only to target the HIV reservoir, but also transverse the site where the reservoir is located.

#### *6.1.1. Cal-1 lentiviral vector*

be expected due to the required production of cells, thus delaying the effect of the therapeutic gene(s). Production of new CD4+ T lymphocytes from the thymus has been predicted to be at a rate of approximately 1.65 cells/μL of blood/day due to thymic function [95]. The resulting in the production of a stable population of protected cells would be expected to gradually

While the modification of HSC has the benefit of long-lasting and broad-spectrum protection via the differentiation of stem cells, this approach still lacks the immediate benefit of targeting the existing CD4+ T cells population. The use of CD4+ T cells as a target for HIV gene therapy has been explored and assessed in several studies. Isolation and modification of CD4+ T cells is relatively simple, as they largely populate and consistently traffic through peripheral blood. Accordingly, no stimulatory factors (such as G-CSF) are required to mobilise them prior to collection. This method has the benefits of providing an immediate benefit via the reintroduction of a protected population of the primary target cells for HIV infection [96]. This has been performed and shown to be both safe in treatment, and effective in delivery of the therapeutic gene [96, 97]. Lentiviral vectors are being increasingly used in clinical trials to treat a variety of diseases ranging from cancers, to genetic diseases such as haemophilia and sickle cell anaemia, as well as several trials treating HIV. The largest such trial in HIV gene therapy demonstrating the safety of lentiviral vectors was the Phase II trial whereby a Tat/Vpr specific anti-HIV ribozyme (OZ1) or placebo was delivered in autologous CD34+ haematopoietic progenitor cells. The trial involved 74 patients where there were no adverse events related to the vectors or infusion

As outlined above, present gene therapy efforts to target HIV are primarily defensive in approach, as they encode future HIV resistance and may not influence the HIV reservoir in the short-term. Given the success of CAR T cell therapy in various cancer trials, many investigators are now multiplexing HIV resistance alongside a CAR construct that can target HIV. As cellular markers for the HIV reservoir are often shared in various leukocyte niches, the equivalent to the anti-CD19 approach used in B cell leukaemia has yet to be determined. Rather, investigators have now turned to potent broadly neutralising antibodies, which have been screened and cloned from various HIV positive patients and target HIV envelope. In this setting, several pre-clinical studies are underway in non-human primates in the laboratories of Kiem and Jerome, where resistance afforded by C46 and shRNA is complexed with one of several CAR modules that incorporates the single variable change of well characterised

In contrast, work led by the Berger laboratory has taken a similar but different approach to CAR T cell development. Rather than incorporating a neutralising antibody, they have complexed the first Ig-Like domains of CD4 with the serum mannose binding lectin [100]. This approach enables global recognition of HIV envelope, as it engages the CD4 binding site and also the abundant glycosylation sites that decorate the antigenic silent face of HIV Env. In the CAR T cell context, CD8+ T cells are generated alongside HIV resistant CD4+ T cells to mediate attack on the HIV reservoir. The only problem with the latter approach is that the major HIV reservoir *in vivo* resides in the germinal centers of secondary lymphoid tissue and actively excludes CD8+ T cells, given they lack the germinal homing receptor CXCR5. Therefore, whilst CD4+ T cells may transverse the germinal center, CD8+ T cells will not.

create a positive impact on CD4+ T cell number and help suppress viral load.

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

process [98].

broadly neutralising antibodies [99].

One vector of note that has been extensively studied is the Cal-1 vector, which uses both the maC46 fusion inhibitor and shRNA-CCR5. This construct has been extensively studied, consistently showing therapeutic benefits *in vitro*. Additionally, this construct has also shown its enhanced efficacy when compared against individual genes, as the effect is induced by the use of two therapeutic targets [102]. This has not only led to stronger protection from HIV infection, but also is likely to result in reduced risk of mutation resistance [102]. This has been examined in mouse studies and non-human primates, where it has shown safety, high levels of engraftment (including in CD34+ cells), and a selective growth advantage [102–105].

The Cal-1 therapeutic construct is currently undergoing Phase I/II clinical trials [106]. The study involves 12 HIV positive patients, which have undergone transduction of both HSC and CD4+ T cells with a lentiviral vector carrying both the shRNA-CCR5 and C46 fusion inhibitor. The patients were divided into 3 equal groups, group 1 received no busulfan preconditioning, group 2 received 4 mg/kg busulfan, and group 3 received 2 doses to a total of 6 mg/kg busulfan conditioning. This study is currently ongoing but will provide important data on the optimised conditioning treatment to guide future treatment studies.

#### *6.1.2. Limitations of lentiviral vector delivery*

Whilst we now have therapeutic approaches that can focus our efforts on a HIV cure, delivery of these components still presents a barrier. Lentiviral vectors have proven to be extremely useful in providing delivery of therapeutic genes, although there are still limitations. As mentioned, cells can only be modified *ex vivo*, thus requiring apheresis. Additionally, in the case of HIV, as ART will prevent uptake of the lentiviral vector, patients must first stop ART, thus raising various health concerns and ethical obstacles. Furthermore, current approved lentiviral platforms can only transduce T cells that are activated, as this over comes lentiviral restrictions at the membrane and underneath the membrane. The sum of these problems significantly increases the cost of the clinical approach. In the setting of CAR-T cells the estimates for treatment of refractory B cell leukaemia is approximately \$US400,000. Given this substantial cost, the accessibility of this type of therapeutic intervention is low. Thus, efforts are underway that will improve the process of this gene delivery pipeline. For instance, lentiviral vectors could be developed to target fresh leukocyte populations *ex vivo*, obviating the need for large scale apheresis. Additionally, lentiviral vectors could be modified to target leukocyte subsets, so the cells with the greatest stem-like attributes are re-infused and not diluted with cells that may not proceed down the differentiation pathway. This could potentially include a sub-population of resting T cells (*e.g.* Stem T cells) being isolated, genetically modified and re-infused in a manner that may not require apheresis. However, whether re-infusion of a smaller population of stem T cell would result in the same outcome that maybe achieved with a large population of bulk T cells obtained by leukopheresis needs to be thoroughly investigated.

#### **6.2.** *In vivo* **delivery by nanoparticles**

The *ex vivo* delivery of an HIV gene therapy treatment will only be achievable in developed countries with the appropriate resources to facilitate the approach and this will not be feasible in countries which currently have the largest burden of HIV, such as sub-Saharan Africa. We and others are working on an alternate and highly relevant systemic, *in vivo* approach, which may ultimately be accessible to all. This approach utilises nanotechnology to deliver the HIV therapeutic to target cells, ideally those of the latent reservoir. According to the Recommendation of the European Commission in 2011 the currently accepted definition of a nanoparticle (NP) is a particle where one or more external dimensions is in the size range of 1 to 100 nm [107]. However, larger particles with sizes up 1000 and 2000 nanometres are commonly referred to as 'nano', especially since for medical purposes the size range of ≤100 nm is not always practical, as a larger surface can carry more drug on a single particle [108, 109]. However, to be able to be used in the human body, NPs must be biocompatible and without cytotoxic side effects [108, 109].

to the gold nanoparticle and involved incorporation of a thiol group to generate thiolated raltegravir. Cellular uptake and toxicity of AuNps was assessed in three different cell types; PBMCs, macrophages and HBMECs and confocal microscopy showed AuNPs inside all three cell types 24 hours post-delivery [113]. No toxicity was observed between 24 and 72 hours post-delivery. Importantly, the study investigated *in vivo* delivery of AuNPs and reported the presence of AuNPs in multiple sites, with the highest to lowest levels observed in spleen, liver, kidney, tail, heart, blood, lungs, muscle and brain of BALB mice 24 hours post-delivery [113]. Accumulation of AuNPs in the spleen and liver was attributed to reticuloendothelial system clearance, which is the bodies first line of defence for any *in vivo* delivered therapy. The lack of a specific marker for the latent reservoir is an ongoing challenge for targeting cells which harbour integrated HIV DNA and have the potential to reactivate and produce productive virus. Although this approach does successfully penetrate some cells of the latent reservoir, *i.e.* lymphocytes and macrophages, and to a modest degree cells in the brain, it is not a targeted approach and will most likely need further development of functional groups to penetrate the majority of cells of the latent reservoir. Due to the rarity of cells harbouring latent provirus, which is estimated to

cells, targeting these cells is the current challenge for an HIV cure.

mice were generated and infected with HIV-1NL4-3 12 weeks

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

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

The cationic PAMAM dendrimer NP system is comprised of highly branched, chemical polymers with cationic primary amine groups on a spherical surface that form stable, uniform nanoscale complexes. The PAMAM dendrimer interacts electrostatically with negatively charged dicer substrate siRNAs (dsiRNAs). The combination of anti-HIV siRNAs in this study included tat/rev, as well as the siRNAs targeting the CD4 and TNP03 genes [114]. In

following engraftment, then dendrimer-siRNA complexes were delivered via *i.v.* injection using equal amounts of all three siRNAs. Injections were continued weekly for 4 weeks. A significant decrease in HIV viral load by 3 logs relative to controls was reported and persisted up to 3 weeks post-treatment, however virus rebound was observed in the majority of animals after this time point [114], as is the standard response in patients following ART cessation. The study then investigated redosing of the dendrimer-siRNA complexes 3 months following the last administration and observed a further virus suppression which persisted for 3 weeks past the additional treatment. Assessment of mRNA levels of the three targeted genes (HIV tat/rev, CD4 and TNPO3) showed reductions in mRNA levels relative to the controls corresponded to the dosing schedule and confirmed sequence-specific and efficient gene silencing [114]. The main challenge with this approach is the need for continual treatment, or alternately the further development of a sustained-release approach. Further, whether this approach will be able to target very rare cells harbouring the latent reservoir remains to be investigated.

The field of HIV gene therapy is rapidly evolving, with development of both novel anti-HIV therapeutics and delivery systems to ensure cell specific targeting. While an *ex vivo* gene therapy approach for HIV is well on the path to patient translation, further targeting of the latent reservoir will be necessary to achieve a systemic, *in vivo* gene therapy approach. This will require identification of biomarker/s for latently-infected cells and novel ways to incorporate them into viral vectors and/or nanoparticle platforms. Once achieved, the next challenge

be 1 in every 10<sup>6</sup>

**7. Conclusion**

this study, humanised Rag2−

/ − γc− / −

Concentrating on HIV drug delivery, NPs have the unique feature of being able to absorb and carry other compounds on their relatively large functional surface [109]. Using NPs as delivery agents has the potential advantages of highly specific and controlled drug delivery to a targeted tissue or cell, such as those of the latent reservoir, keeping non-target organs and cells free of the drug, thereby reducing toxicity. Further, by releasing the drug in a controlled manner at a predetermined rate, achieved through changes in the physiological environment like pH, temperature or enzymatic activity, the resulting therapeutic efficacy can be increased [108–110]. Importantly, nano-based delivery systems have been shown to transport therapeutics across the blood-brain barrier, which is highly relevant for treating neuro-degenerative diseases and specifically the HIV reservoir in the central nervous system [111]. Prior to use of NPs in humans, the following basic prerequisites need to be known: drug incorporation and release, formulation stability and shelf life, biocompatibility, biodistribution and targeting, potential toxicities as well as functionality [109]. Another consideration are the possible adverse effects of residual material after drug delivery, therefore biodegradable NPs with a limited life span are optimal [109].

There are many types of NPs reported as delivery vehicles for HIV therapeutics, such as liposomes, micelles, polymer capsules, inorganic gold particles and dendrimers [112]. The number of different formulations of NPs being explored for HIV and other diseases is steadily increasing and a focused review on nanoparticle systems is provided by Pelaz et al. [112]. An example of the *in vivo* gene therapy process is depicted in **Figure 4**.

In the case of HIV, NPs have been used to deliver antiretroviral drugs or anti-HIV therapeutics, such as siRNAs. Inorganic gold particles delivering antiretrovirals have progressed through to *in vivo* delivery in mouse models, as have poly(amidoamine) PAMAM dendrimers and RNA-aptamer conjugates (as previously describes in Section 3.2.2.2), that deliver a combination of anti-HIV siRNAs. The gold particles and PAMAM dendrimer nano-platforms will be discussed below to highlight the challenges of targeting the HIV latent reservoir.

The NP platforms delivering an antiretroviral drug were comprised of inorganic gold nanoparticles particles (AuNPs) ~2–10 nanometers in diameter and were conjugated with an HIV integrase inhibitor, raltegravir [113]. Modification of raltegravir was necessary to link the inhibitor to the gold nanoparticle and involved incorporation of a thiol group to generate thiolated raltegravir. Cellular uptake and toxicity of AuNps was assessed in three different cell types; PBMCs, macrophages and HBMECs and confocal microscopy showed AuNPs inside all three cell types 24 hours post-delivery [113]. No toxicity was observed between 24 and 72 hours post-delivery. Importantly, the study investigated *in vivo* delivery of AuNPs and reported the presence of AuNPs in multiple sites, with the highest to lowest levels observed in spleen, liver, kidney, tail, heart, blood, lungs, muscle and brain of BALB mice 24 hours post-delivery [113]. Accumulation of AuNPs in the spleen and liver was attributed to reticuloendothelial system clearance, which is the bodies first line of defence for any *in vivo* delivered therapy. The lack of a specific marker for the latent reservoir is an ongoing challenge for targeting cells which harbour integrated HIV DNA and have the potential to reactivate and produce productive virus. Although this approach does successfully penetrate some cells of the latent reservoir, *i.e.* lymphocytes and macrophages, and to a modest degree cells in the brain, it is not a targeted approach and will most likely need further development of functional groups to penetrate the majority of cells of the latent reservoir. Due to the rarity of cells harbouring latent provirus, which is estimated to be 1 in every 10<sup>6</sup> cells, targeting these cells is the current challenge for an HIV cure.

The cationic PAMAM dendrimer NP system is comprised of highly branched, chemical polymers with cationic primary amine groups on a spherical surface that form stable, uniform nanoscale complexes. The PAMAM dendrimer interacts electrostatically with negatively charged dicer substrate siRNAs (dsiRNAs). The combination of anti-HIV siRNAs in this study included tat/rev, as well as the siRNAs targeting the CD4 and TNP03 genes [114]. In this study, humanised Rag2− / − γc− / − mice were generated and infected with HIV-1NL4-3 12 weeks following engraftment, then dendrimer-siRNA complexes were delivered via *i.v.* injection using equal amounts of all three siRNAs. Injections were continued weekly for 4 weeks. A significant decrease in HIV viral load by 3 logs relative to controls was reported and persisted up to 3 weeks post-treatment, however virus rebound was observed in the majority of animals after this time point [114], as is the standard response in patients following ART cessation. The study then investigated redosing of the dendrimer-siRNA complexes 3 months following the last administration and observed a further virus suppression which persisted for 3 weeks past the additional treatment. Assessment of mRNA levels of the three targeted genes (HIV tat/rev, CD4 and TNPO3) showed reductions in mRNA levels relative to the controls corresponded to the dosing schedule and confirmed sequence-specific and efficient gene silencing [114]. The main challenge with this approach is the need for continual treatment, or alternately the further development of a sustained-release approach. Further, whether this approach will be able to target very rare cells harbouring the latent reservoir remains to be investigated.
