The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS

Oligonucleotide therapeutics

original article

© The American Society of Gene & Cell Therapy

The Impact of Unprotected T Cells in RNAi-based Gene Therapy for HIV-AIDS Elena Herrera-Carrillo1, Ying Poi Liu1 and Ben Berkhout1 1 Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

RNA interference (RNAi) is highly effective in inhibiting human immunodeficiency virus type 1 (HIV-1) replication by the expression of antiviral short hairpin RNA (shRNA) in stably transduced T-cell lines. For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs against highly conserved regions of the HIV-1 RNA genome. The future in vivo application of such a gene therapy protocol will reach only a fraction of the T cells, such that HIV-1 replication will continue in the unmodified T cells, thereby possibly frustrating the therapy by generation of HIV-1 variants that escape from the inhibition imposed by the protected cells. We studied virus inhibition and evolution in pure cultures of shRNA-expressing cells versus mixed cell cultures of protected and unprotected T cells. The addition of the unprotected T cells indeed seems to accelerate HIV-1 evolution and escape from a single shRNA inhibitor. However, expression of three antiviral shRNAs from a single lentiviral vector prevents virus escape even in the presence of unprotected cells. These results support the idea to validate the therapeutic potential of this anti-HIV approach in appropriate in vivo models. Received 12 August 2013; accepted 1 December 2013; advance online publication 4 February 2014. doi:10.1038/mt.2013.280

INTRODUCTION

The etiologic agent of the acquired immunodeficiency syndrome (AIDS) is the human immunodeficiency virus type 1 (HIV-1). This pandemic infection affects millions of people worldwide. The only effective way to currently treat HIV is a highly active antiretroviral therapy that combines multiple antiretroviral drugs to prevent the emergence of drug-resistant strains. Highly active antiretroviral therapy usually combines at least three drugs, including reverse transcriptase (RT), protease, fusion/entry, or integrase (IN) inhibitors. The development of drugs that target different steps of the HIV-1 replication cycle is important to avoid cross-resistance such that viral escape requires mutations in multiple drug targets.1 Highly active antiretroviral therapy can effectively control viral replication, but it fails to achieve complete viral clearance.2 Furthermore, the emergence of multiple drug-resistant HIV-1 strains–related to the extreme mutability of the virus–and the side effects of drug-based therapies urge the development of

alternative approaches for treatment of HIV infection.3–5 RNAbased antiviral approaches are among the most promising for development of durable anti-HIV therapies.6–9 The cellular RNA interference (RNAi) mechanism triggers the processing of noncoding microRNAs that regulate gene expression at the posttranscriptional level to control cell differentiation and development.10 This pathway can also be induced by artificial short hairpin RNAs (shRNAs) that are produced in the cell from an introduced transgene and processed into small interfering RNAs (siRNAs).11 Lentiviral vectors provide a means to stably express shRNA from the integrated vector to induce stable and long-term gene silencing in both dividing and nondividing cells.12,13 Thus, in less than a decade after the discovery of RNAi-mediated gene silencing, it has already been tested as potential HIV-1 therapy in clinical trials.14 Although RNAi can inhibit HIV-1 replication effectively, a single nucleotide change in nearly any position of the 19-nucleotide target sequence is sufficient to generate shRNAresistant viruses during prolonged virus passage, even in highly conserved target sequences.15–17 For the development of a durable gene therapy that prevents viral escape, we proposed to combine multiple shRNAs that target highly conserved HIV-1 regions. Thus, the goal is to use a lentiviral vector with multiple shRNA cassettes that becomes stably incorporated in the human genome.18 Safety and efficacy studies can be performed in the “Human Immune System” mouse model.19,20 Human CD34+ hematopoietic progenitor cells are transduced ex vivo with the lentiviral RNAi expression constructs and injected into immunocompromised newborn mice to monitor cell development and differentiation, shRNA expression, cytotoxicity, and efficacy of the therapeutic regimen on HIV-1 infection. This preclinical animal model does closely mimic the anti-HIV gene therapy approach proposed for HIV-infected patients.21 We tested the four candidate shRNAs and observed normal development of the human immune system and no adverse effects in a competitive cell growth assay for three shRNAs. A negative impact of a single shRNA on in vitro cell growth and hematopoiesis in the human immune system mouse was observed, which led to its removal from the translational track toward a clinical trial.22,23 We, therefore, created the combinatorial RNAi vector R3A that expresses the three nontoxic and potent antiviral shRNAs: Pol1, Pol47, and RT5. We previously investigated the efficacy of the combinatorial RNAi vector R3A in pure cultures of shRNA-expressing cells.24

Correspondence: Ben Berkhout, Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam, Academic Medical Center of the University of Amsterdam, K3-110, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected]

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© The American Society of Gene & Cell Therapy

However, the in vivo application in an already infected HIV-1 patient is likely to be more complex. For instance, no matter whether the gene therapy will target the primary HIV-1 target cells, the CD4+ T cells, or the CD34+ hematopoietic precursor cells that reside in the bone marrow and that will provide a continuous source of mature immune cells including T cells, only a fraction of these cells will become modified and protected. Thus, virus replication may continue in T cells that are not modified by the gene therapy. These cells will be removed rapidly by the immune system on presentation of viral antigens on the cell surface, but new unmodified cells will soon arrive in the periphery. Ongoing virus replication in unmodified cells may generate a mixture of virus variants or quasispecies over time. This quasispecies could thus enhance the appearance of candidate escape variants that will be selected on passage in the modified shRNA-expressing cells. In this study, we tested the inhibitory effect of a single shRNA and the combinatorial shRNA gene therapy in cultures with different ratios of protected/unprotected cells. The possible enhancement of unprotected cells on the evolution of HIV-1 variants that are resistant to shRNA-mediated inhibition is illustrated in Figure 1.

Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

In vitro

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RESULTS

Over the years, many antiviral shRNA inhibitors have been evaluated in our laboratory.1,25 We intentionally chose shRNAs that target HIV-1 RNA sequences that are relatively conserved among virus isolates and even distinct subtypes. For this study, we selected the individual shRNA Pol47 and the combinatorial shRNA construct R3A, which expresses three shRNAs simultaneously (Pol1, Pol47, and RT5). R3A achieves durable inhibition by raising the genetic threshold for viral escape.26–28 The different shRNA expression cassettes were cloned into the lentiviral vector JS1 to allow stable transduction of T cells. The previous screening of shRNAs was performed in the SupT1 T-cell line, a commonly used CD4+ T-cell line that expresses the CXCR4 (X4) co-receptor and that is permissive for HIV-1 infection, showing clear cytopathic effects (syncytia) on virus replication. We now included the PM1 T-cell line that constitutively expresses both CXCR4 and CCR5 (R5) co-receptors.29 This cell line will allow us to test R5-using viruses in the future, but was chosen for this study because it does not form large multicell syncytia on HIV-1 infection. This seems an important measure to avoid mixing of modified and unmodified cells. Nevertheless, some cytopathic effects can be observed with X4-using HIV-1 isolates in PM1 cells (unpublished results). The lentiviral transduction of SupT1 and PM1 T-cell lines was performed at a multiplicity of infection of 0.15 to obtain maximally a single integrated lentiviral vector per cell to avoid shRNA overexpression and saturation of the RNAi machinery. Green fluorescent protein (GFP) is encoded by the vector, and GFP-positive cells were selected for virus infection studies.

Antiviral activity in stably transduced cells and escape studies Several scenarios were considered in the experimental design of viral escape studies with mixed cell cultures. Virus evolution is usually studied in pure cultures of shRNA-expressing cells (blue) that are protected against HIV-1 infection (Figure 1, in vitro panel). Variants will be generated at a low rate because of the Molecular Therapy  vol. 22 no. 3 mar. 2014

Unmodified cells (unprotected)

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Phenotyping

Standard genotyping

Figure 1 Human immunodeficiency virus type 1 (HIV-1) escape from short hairpin RNA (shRNA) therapy in different culture systems. In vitro panel: pure cultures of shRNA-expressing cells (blue) that are protected against HIV-1 infection. HIV-1 variants will be generated at a low rate and only a shRNA-resistant variant (red virus) will be able to spread. In vivo context: mixed cell cultures of protected (blue) and unprotected (pink) cells will allow virus replication in the unprotected cells, leading to the rapid generation of a viral quasispecies by spontaneously acquired mutations. This quasispecies may also contain one or more shRNA-resistant variants that are able to replicate in the protected cells. Test panel: direct genotyping means sequencing of proviral DNA target sequences when virus replication is observed in the primary virus cultures. Phenotyping: cell-free virus harvested in the primary culture is used to produce a replication curve on unprotected cells and shRNAexpressing cells. Standard genotyping of proviral DNA target sequences when virus replication is observed in the latter shRNA-expressing cells. WT, wild-type.

severe, but not absolute block of virus replication. Once a shRNAresistant HIV-1 variant (red virus) is generated, it will replicate and spread in these cells. We used mixed cell cultures of protected and unprotected cells to study HIV-1 evolution under conditions that mimic those in a treated patient (Figure 1, in vivo context). Virus replication will occur unhindered in the unprotected cells (passage through pink cells), leading to the rapid generation of a viral quasispecies over time by spontaneously acquired mutations. This quasispecies may include shRNA-resistant variants that are uniquely able to replicate in the protected cells. Resistant HIV-1 variants could also be generated in the protected cells, but at a greatly reduced rate due 597

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Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

the viruses that caused the peak in CA-p24 values represent true escape variants or the input wild-type (WT) virus. To do so, we first analyzed the sequence of the shRNA target sequence to document viral escape. This direct genotypic test was performed by polymerase chain reaction (PCR) amplification of the targeted HIV-1 genome sequences as part of the integrated proviruses established in primary culture at the peak of breakthrough replication as shown in Figure 2. The results indicate mutational escape in all cultures without and with 5% unprotected cells (Table 1). The 20% unprotected culture yielded a mutant genotype in three of six cultures and no escape was apparent in the 50% cultures. An initial conclusion may thus be that accelerated evolution and Pol47 escape is apparent in the presence of 5 and 20% unprotected cells. These initial results confirm the notion that only a small subset of mutations–mostly silent codon changes that do not affect the encoded integrase enzyme (last column in Table 1)–are allowed in these highly conserved HIV-1 sequences, exactly as reported previously.17 For instance, the silent G8A mutation was scored in 10 independent cultures. Position G12 was mutated in a nonsilent manner in four cultures (2× G12A, 1× G12C, and 1× G12U). A double mutation in the target sequence was scored in two cultures (5-2 and 20-2). We previously tested such mutated virus variants for replication characteristics.17,30 Only WT virus was apparent in the cultures with 50% unprotected cells and the 100% unprotected control. Of course this population sequence analysis (direct genotyping) will only detect the majority of virus variants in a complex virus mixture (illustrated in Figure 1). Importantly, some of the HIV-1 variants generated by passage on the unprotected cells may have the shRNA-resistant phenotype that allows their expansion on the restricted shRNA-expressing cells, but they will likely remain minor species in this mixed cell culture system. To detect those minority variants, we performed specific additional experiments. We performed a phenotypic test for the presence of shRNAresistant HIV-1 variants by passage of the cell-free virus on pure SupT1-shRNA cells (an example is presented in Figure 3). This should filter out the nonresistant virus variants, including WT

to shRNA-mediated suppression of virus replication. Besides the difference in timing of HIV-1 escape, the outcome of these two evolution scenarios will also be strikingly different. The shRNAresistant HIV-1 variants will dominate the in vitro culture conditions, but remain minority variants in the in vivo situation. To test the impact of unprotected cells on HIV-1 evolution, we challenged SupT1-shRNA and PM1-shRNA cells with the X4-using primary isolate HIV-1 LAI at different ratios of the respective protected and unprotected cells (5, 20, and 50% unprotected cells). We realize that this may not ideally mimic the in vivo context of a future gene therapy, but adding even more unprotected cells will lead to unhindered virus replication. We will compare the evolution results with those obtained in pure cultures with only shRNA-expressing cells. The empty lentiviral vector (JS1) was used as negative control. We will describe the SupT1shRNA experiments in much detail and will refer to the PM1 cell experiments in more general terms. We will first describe the results for the single shRNA inhibitor (Pol47).

The impact of unprotected cells on the evolution of Pol47 resistance We added 5, 20, and 50% unprotected T cells to regular SupT1Pol47 cultures, followed by infection with the HIV-1 LAI isolate at a multiplicity of infection of 0.002. We split the cell mixture when needed and monitored for viral spread by performing CA-p24 enzyme-linked immunosorbent assay on the culture supernatant samples (Figure 2). We performed six parallel cultures per experimental condition because virus evolution is a chance process. It took about 31–55 days for viral escape to occur in the pure SupT1shRNA cells that served as control culture. As predicted, the gradual addition of unprotected cells does cause a shift toward more rapid viral “escape.” Escape accelerated from an average of 48 days for pure SupT1-shRNA cultures to around 33, 21, and 15 days with 5, 20, and 50% unprotected cells, respectively. However, we obviously cannot exclude that this “escape” represents the input LAI virus that starts spreading in the unprotected cells when present at a certain percentage. Thus, it should be validated whether SupT1- Pol47

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Figure 2 Impact of unprotected cells on the evolution of Pol47 resistance. A control vector transduced cell line was included (100% unprotected cells) and 5, 20, and 50% of unprotected cells was added to regular SupT1-Pol47 cultures to compare the evolution results with those obtained in pure cultures of Pol47-expressing cells. Six parallel cultures per experimental condition were challenged with human immunodeficiency virus type 1 LAI at a multiplicity of infection of 0.002. Virus replication was monitored by measuring CA-p24 for 55 days.

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AA, amino acid. a Percentage of unprotected cells in the culture. bNumber of the culture. cDay of viral escape. dThe Pol47 target position 1–19, white bars indicate integrase codons. eAA substitutions in the integrase enzyme. f Shadowed nucleotide changes indicate a nonpure sequence: the mutation is most abundant, but minority sequence(s) are present (either WT or other mutations).

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Integrase positiond

Table 1 Direct genotyping of the proviral target sequence on SupT1-Pol47 infected cultures

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Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

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Figure 3 Phenotypic test of emerged LAI viruses. Cell-free virus at the peak of virus production in every scenario described (5, 20, and 50% unprotected cells and pure cultures of Pol47-expressing cells) was collected and passaged on (a) fresh unprotected cells and (b) SupT1-Pol47 cells (pure cultures of protected cells). Wild-type (WT) virus was included as control (virus that replicates exclusively on unprotected cells).

HIV-1, and select the preexisting shRNA-resistant variants. Whereas the initial genotyping may also detect archived early (WT) viruses, this second genotyping on reculture will focus on the freshly integrated proviruses. In fact, all SupT1-Pol47 cultures did show the efficient spread of Pol47-resistant virus, also for the three 20% cultures and all 50% cultures that did not reveal any resistance mutation in direct genotyping. A second genotype analysis was performed on the cultures that became positive, which confirmed the presence of resistance mutations (Table 2). Again a preponderance of the G8A mutation is scored in 18 of 24 cultures. Thus, resistant virus was generated in all evolution scenarios, including the 50% cultures, but these variants could be detected only on passage on restricted SupT1-Pol47 cells. These combined results demonstrate that unprotected cells can significantly accelerate the evolution of single shRNA resistance. Besides shortening of the time frame for generation of shRNA-resistant variants, the presence of unprotected cells also seems to accelerate virus evolution in another way. We detected the presence of multiple mutations in the mixed cultures and not in the pure shRNA cell cultures (compare Tables 2 and 1, respectively). Double mutants were observed in five mixed cultures (Table 2, cultures 5-2, 20-1, 20-2, 20-5, and 50-3). These results are even more remarkable with respect to the shorter evolution time needed for the mixed cultures (e.g., around 15 days for 50% unprotected cells compared with an average of 48 days for pure SupT1-shRNA cells). However, there is one technical complication. The population sequencing does not allow one to discriminate between two possibilities: the presence of a true double mutant or the simultaneous presence of two single mutants. We performed clonal sequencing to solve this. The PCR products were cloned, transformed into bacteria, and 12 individual bacterial clones were sequenced for each of these five cultures (Table 3). The results clearly indicate the coexistence of two single mutants in these cultures, but sometimes a more complex evolutionary scenario was observed. For instance, culture 20-2 yielded three different single mutants (G8A, G12A, and G15A) and two double mutants (G6A-G12A and G9A-G15A) that were derived from some of the available single mutants, most likely by recombination. In fact, the simultaneous appearance of multiple escape variants may also be a sign of accelerated viral escape. 600

We performed similar HIV-1 evolution studies using the PM1 T-cell line, with shRNA-expressing cells and an increasing percentage of unprotected cells, again showing that unprotected cells accelerate viral escape. The follow-up test (direct genotype and phenotype + genotype) confirmed the results obtained in SupT1 cells. We used all experimental data in the subsequent analysis of the mutational patterns (see below).

The impact of unprotected cells on the evolution of R3A resistance The standard selection protocol on pure SupT1-R3A cultures fails to yield any resistant virus in the six cultures tested (Figure 4). By adding unprotected cells, we did start to detect replicating virus in the culture with 20 and 50% unprotected cells, but of course the immediate question arises whether this simply reflects the WT virus replicating on unprotected cells. HIV-1 replication was only delayed by a few days in the mixed cultures compared with the positive control culture with 100% unprotected cells. To test whether R3A-resistant virus was selected in the 20 and 50% mixed cultures, we performed the phenotype test by passage on pure SupT1-R3A cells in sixfold. For this, we used a relatively low and high input (0.1 and 5 ng of CA-p24, respectively). No viral escape was apparent in any of the 12 low-dose passages (6 × 20 and 6 × 50%, no syncytia visible, no CA-p24 measured in the culture supernatant after prolonged cultures). At the high viral input, three of the 20% cultures became positive (syncytia and CA-p24), but this may represent “breakthrough replication” that we described previously when the inhibitor is overwhelmed by a high virus input.18 To proof that these positive cultures do not represent true R3A escape viruses, we performed two additional tests. First, we analyzed the HIV-1 genotype, but did not detect any resistance-causing mutations in the three R3A target sequences by population-based sequencing. Second, we passaged the three positive samples onto restricted SupT1-R3A cells, but there was no viral replication detectable (no syncytia visible and not CA-p24 in the culture supernatant). To test whether partial resistance had occurred, i.e., resistance to one or two of the triple shRNAs, we also passaged the positive samples onto the respective single shRNA cells, but never detected any virus replication (results not shown). Thus, we found no evidence for R3A-escape, www.moleculartherapy.org  vol. 22 no. 3 mar. 2014

Molecular Therapy  vol. 22 no. 3 mar. 2014

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integrase

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See Table 1 for further details. Shadowed nucleotide changes indicate a nonpure sequence: the mutation is most abundant, but minority sequence(s) are present (either WT or other mutations). AA, amino acid.

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Integrase position

Table 2 Standard genotyping of the proviral target sequence on SupT1-Pol47 infected cultures

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© The American Society of Gene & Cell Therapy

Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

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AA, amino acid. a Percentage of unprotected cells in the culture. bNumber of the culture. cNumber of cultures with this particular sequence (total = 12). d The Pol47 target position 1–19, white bars indicate integrase codons. eAA substitutions in the integrase enzyme.

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Table 3 Clonal sequencing of the proviral target sequence of SupT1-Pol47 infected cultures

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Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS © The American Society of Gene & Cell Therapy

www.moleculartherapy.org  vol. 22 no. 3 mar. 2014

© The American Society of Gene & Cell Therapy

Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

10,000

SupT1- R3A

CA-p24 (ng/ml)

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Percent of unprotected cells 5% 20%

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0.1

R3A - shRNA cells 0.01

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Days postinfection

Figure 4 Impact of unprotected cells on the evolution of R3A resistance. A control vector transduced cell line was included (100% unprotected cells) and 5, 20, and 50% of unprotected cells were added to regular SupT1-R3A. Six parallel cultures per experimental condition were challenged with human immunodeficiency virus type 1 LAI at a multiplicity of infection of 0.002. Virus replication was monitored by measuring CA-p24 for 70 days.

not even partial escape from one of the shRNA inhibitors, due to the presence of helper unprotected cells.

Positional hotspots within the Pol47 target sequence for viral escape Based on the large number of shRNA Pol47 escape cultures analyzed in the course of this study, we confirmed the preferential use of certain viral escape paths.17 These results are plotted in Figure 5 for the combined SupT1 and PM1 experiments. The same mutational preference was observed in these two T-cell lines (Figure 5a,b). We also scored the type of mutation that underlie RNAi escape, for which we compiled the SupT1 and PM1 data (Figure 5c). The most favorite escape mutation occurs at silent codon position 8 (57% of all evolution cultures), followed by the nonsilent codon positions 12 (25.9%), 15 (7.4%), 9 (5.6%), and 10 (1.9%) and the silent position 17 (1.9%). There may be several reasons for the preponderance of the G8A mutation. First, it is in the center of the shRNA target sequence, which may be beneficial for the escape phenotype by disrupting the center of the siRNAmRNA duplex. Second, it represents a silent codon change (GGGto-GGA, both encoding Gly), and thus the WT integrase enzyme is produced without an impact on viral replication fitness. In fact, the absence of the alternative silent codon changes at this position (GGU and GGC, both also encoding Gly) does point to another major determinant of evolution: the mutational bias that strongly favors transitions over transversions with a preponderance of G-to-A changes.31–36 We found 94.4% of transversions versus 5.6% of transitions and a preponderance of G-to-A changes to underlie Pol47 escape (91% of all mutations observed). It is striking that, in addition to the G8A hotspot, only a single silent codon mutation was selected under RNAi pressure (A17G). The other possible silent codon positions (2, 5, 11, and 14) were not selected, probably because they do not provide a high level of RNAi resistance. We also observed that conservative amino acid substitutions (Ala-to-Val, Val-to-Leu/Ile, and Val-to-Ile) are predominant among nonsilent escape mutations. All other possible sequence changes are likely not selected because of suboptimal Molecular Therapy  vol. 22 no. 3 mar. 2014

RNAi resistance and/or decreased activity of the mutant integrase. Thus, the virus uses relatively few of the theoretically possible escape routes, most likely as strategy to maintain a certain level of replication fitness.

DISCUSSION

RNA viruses have a high propensity to modify their genomes and acquire resistance to antiviral drugs or siRNAs because of the high error rate of viral RNA-dependent RNA polymerases, and the same is true for the RT enzyme of HIV-1.37,38 Many studies have shown that HIV-1 and other viruses can escape from RNAi pressure by a single mutation in the siRNA target sequence despite the fact that highly conserved viral genome sequences were targeted.15–17,39,40 Thus, the development of a combinatorial RNAi approach that targets different steps of the HIV-1 replication cycle remains important because it requires the acquisition of mutations in multiple siRNA target sites. We designed a combinatorial RNAi approach that comprises three shRNAs targeting three distinct and highly conserved regions of the HIV-1 RNA genome. We showed that the combinatorial approach can provide enhanced and durable suppression of HIV-1 replication in in vitro cultures with a pure population of shRNA-expressing cells. However, the in vivo application in an already infected HIV-1 patient is likely to be more complex. No matter whether the gene therapy targets the CD4+ T cells or the CD34+ hematopoietic precursor cells, only a fraction of HIV-1 target cells will become modified and protected. Although unprotected cells will be removed rapidly by the immune system on HIV-1 infection due to presentation of viral antigens on the cell surface, new unprotected cells will soon arrive in the periphery from the bone marrow where the hematopoietic stem cells reside. Therefore, a low, but constant level of HIV-1 replication will occur in the unmodified cells that may generate a mixture of virus variants or quasispecies over time. This is a major concern for future HIV gene therapy clinical trials. In this study, we attempted to explore the impact of unprotected T cells on HIV-1 evolution in the face of an effective RNAi gene therapy. We first produced stably T-cell lines (SupT1 and PM1) expressing 603

© The American Society of Gene & Cell Therapy

Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

a

b *

60

Relative distribution (%)

Relative distribution (%)

80

40 Leu Leu

20

lle

Thr Val 0

lle

80

A G U

60

40 Leu lle

20

lle Thr

* 0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 G G U G A A G G G G C A G U A G U A A U A Gly Glu Gly Ala Val Val lle

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 G G U G A A G G G G C A G U A G U A A U A Glu Val lle Gly Gly Ala Val

Target nucleotide position

Target nucleotide position

c

C

*

80

Relative distribution (%)

* 60

40 Leu Leu

20 Thr

lle Val

0

lle

*

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 G G U G A A G G G G C A G U A G U A A U A Gly Glu Gly Ala Val Val lle Target nucleotide position

Figure 5 Distribution of escape mutations within the 19-nucleotide target sequence (Pol47 shRNA escape). (a) Composite of mutations observed within the 19-nucleotide target in multiple independent viral escape cultures in SupT1 and (b) in PM1 T-cell line. (c) Composite of mutations observed in both T-cell lines (SupT1 + PM1). *Silent mutation. shRNA, short hairpin RNA.

a single shRNA Pol47 or the combinatorial shRNA candidate R3A. We sorted GFP+ cells to have pure cultures of protected cells and we added back 5, 20, and 50% unprotected cells to study their impact on HIV-1 escape possibilities. We challenged the different cultures with the clonal HIV-1 LAI isolate and monitored viral spread and evolution over time. For Pol47-expressing cells, the gradual addition of unprotected cells caused a shift toward more rapid viral escape from an average of 48 days for pure cultures to around 33, 21, and 15 days with 5, 20, and 50% unprotected cells, respectively. We analyzed the sequence of the HIV-1 target site to document true viral escape because the addition of unprotected cells may also support “false” replication of WT virus. Mutations were detected in the Pol47 target sequence of all escape cultures (pure and mixed cultures), demonstrating that the RNAi inhibition is sequence specific, but more importantly that unprotected cells can accelerate the evolution of single shRNA resistance. We also detected the appearance of double mutants in the target site in the presence of unprotected cells besides shortening of the time frame for generation of shRNA-resistant variants. 604

For R3A-expressing cells, we started to detect replicating virus in the 20 and 50% cell mixtures. To determine whether WT virus is replicating on the added unprotected cells or whether a true R3A-resistant virus was selected, we passaged cell-free virus from the peak of the viral production on pure R3A-expressing cells but also on cells that encode one of the three single shRNAs that comprise R3A (Pol1, Pol47, and RT5) and performed additional tests. No true viral escape as demonstrated by a resistance-causing mutation was detected in any of the experiments. Thus, we demonstrated that the addition of unprotected helper cells does accelerate virus evolution leading to Pol47 resistance, but we found no evidence for R3A escape. These results support the idea to validate the therapeutic potential of this combinatorial RNAi approach in an appropriate in vivo model. One other study did test the impact of unprotected cells on RNAi therapy and viral escape.41,42 This study did in fact report unusual HIV-1 escape routes by mutations in the long terminal repeat promoter and not in the actual shRNA target sites. However, we have argued that this result may be due to the ongoing replication of WT virus in unprotected cells.41 This is exactly www.moleculartherapy.org  vol. 22 no. 3 mar. 2014

© The American Society of Gene & Cell Therapy

what we observed in this study. The reported long terminal repeat mutations are in fact changes that have previously been observed in several unrelated HIV-1 evolution studies in the absence of any RNAi pressure.43,44 To avoid this problem, we probed here the raw material of virus evolution in the mixed cell cultures by passaging on pure shRNAi-expressing cells. This additional step filters out any nonresistant HIV-1 variants. For the Pol47 shRNA inhibitor, we demonstrate the importance of this additional step: no target site mutations were apparent in the raw material of cultures mixed with 50% of unprotected cells, but the truly resistant HIV-1 variants with mutated Pol47 target sequences were present after the additional culture step. These results highlight the many intricacies of these complex virus evolution studies.45,46 More importantly, we confirm that there is no mechanism of resistance against the combinatorial RNAi therapy of the R3A regimen, which renders this combination truly effective. Based on these promising in vitro results, the therapeutic potential of this combinatorial RNAi approach should be tested in appropriate in vivo models to prepare for a clinical trial in humans.

MATERIALS AND METHODS

Plasmid construction. The shRNA expression plasmids based on pSUPER-shRNA were constructed as previously described.11 Lentiviral vector plasmids were derived from the construct pRRLcpptpgkgfppreSsin, which we renamed JS1.47 Expression cassettes for shRNAs were obtained by digestion of pSUPER constructs with XhoI and PstI and inserting this fragment into the corresponding sites of JS1, to create JS1-shRNA. The single shRNA Pol47 and triple shRNA-expressing lentiviral vector R3A were constructed using a third-generation self-inactivating lentiviral vector with GFP reporter as previously described.1 The position of the target sequence on the HXB2 genome and the shRNA sequence is as follows: Pol1 (2328) ACAGGAGCAGAUGAUACAG; Pol47 (4963) GUGAAGGGGCAGUAGUAAU; RT5 (5970) AUGGCAGG AAGAAGCGGAG. These target sequences are highly conserved among HIV-1 isolates, with 100% identity in at least 75% of the 170 complete HIV-1 genomes, including all HIV-1 subtypes, present in the Los Alamos National Laboratory database (www.hiv.lanl.gov). The pLAI plasmid encoding the primary isolate LAI was used to study inhibition of HIV-1 production.48 All DNA constructs were sequence verified using the BigDye terminator cycle sequencing kit (ABI, Foster City, CA). Hairpin RNA constructs were sequenced using a sample denaturation temperature of 98 °C and on addition of 1 mol/l betaine. Cell culture. Human embryonic kidney 293T adherent cells were grown

in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal calf serum (Hybond, Escondido, CA), penicillin (100 U/ml), streptomycin (100 μg/ml), and minimal essential medium nonessential amino acids (Dulbecco’s modified Eagle’s medium/10% fetal calf serum) in a humidified chamber at 37 °C and 5% CO2. SupT1 and PM1 suspension cells were grown in Advanced RPMI (Gibco BRL, Carlsbad, CA) supplemented with l-glutamine, 1% fetal calf serum, penicillin (30 U/ml), and streptomycin (30 μg/ml) at 37 °C and 5% CO2. Lentiviral vector production and transduction. The lentiviral vector was

produced and titrated as previously described.1,49 Briefly, the vector was produced by co-transfection of lentiviral vector plasmid and packaging plasmids pSYNGP, pRSV-rev, and pVSV-g with Lipofectamine 2000 (Invitrogen). After transfection, the medium was replaced with OptiMEM (Invitrogen). The lentiviral vector containing supernatant was collected, filtered (0.45 µm), and aliquots were stored at −80 °C. The transduction titer was measured via GFP expression. SupT1 and PM1 cells were transduced at a multiplicity of infection of 0.15 in a T25 flask seeded with 1 × 106 Molecular Therapy  vol. 22 no. 3 mar. 2014

Impact of Unprotected T Cells in RNAi-based Gene Therapy for AIDS

cells in a total volume of 5 ml to which the lentiviral vector was added. Two days after transduction, live cells were sorted with FACS, and GFP-positive cells were selected. HIV-1 infection. The HIV-1 LAI stock was produced by transfection of

human embryonic kidney 293T cells with the pLAI molecular clone. Cellfree viral stocks were passed through 0.2-µm pore-size filters and titrated in SupT1 and PM1 T cells, measuring virus production by CA-p24 enzymelinked immunosorbent assay (in-house assay). SupT1 and PM1 T cells (3-ml cultures in 6-well plates, 1 × 106 cells/well) were challenged with HIV-1 LAI at the same multiplicity of infection of 0.002. Virus spread was monitored by measuring CA-p24 production (SupT1 and PM1) and scoring of syncytia formation (SupT1) every 2 days. Cells were passaged twice a week.

Direct genotypic test. When virus replication was observed after infec-

tion with HIV-1 LAI, cellular DNA of the infected cells with the integrated provirus was isolated as previously described.50 Integrated proviral DNA sequences were PCR amplified with the following primer pairs (5′-3, the position within pLAI is indicated): Pol1, sense (GTCAGAGCAGA CCAGAGCCAACAG; position 2183) and antisense (GATATTTCTCA TGTTCATCTTGGGCCTTATCTATTCC; position 2659); Pol47, sense (GGCAACTAGATTGTACACATTTAGAAGG; position 4499) and antisense (CTCTTTTTCCTCCATTCTATGGAGA; position 5377); and RT5 sense (ATATCAAGCAGGACATAACAAGG; position 5525) and antisense (TGCTTTAGCATCTGATGCACAAAATA; position 6458) with 30 cycles (1 minute of denaturation at 96 °C, 1 minute of annealing at 62 °C, and 2 minutes of extension at 72 °C). The PCR products were sequenced with the Big Dye Terminator Cycle Sequencing kit (ABI) using the same primers.

Standard genotypic test. When virus replication was observed after infec-

tion with HIV-1 LAI, cell-free virus was passaged to uninfected control and/or shRNA-expressing cells, and virus replication was monitored (phenotyping) (Figure 1, test panel). When virus replication was observed in restricted shRNA cells, cellular DNA of the infected cells was isolated. Integrated proviral DNA sequences were PCR amplified with the primer pairs described above, and the PCR products were sequenced. In case of putative double mutants, the PCR products obtained were gel purified and cloned into the pCR2.1 TOPO vector, which was transformed into competent cells and plated on selective lysogeny/Luria broth plates with ampicillin. Individual bacterial colonies were subsequently sequenced with the T7 or M13R primers.

ACKNOWLEDGMENTS We thank Stephan Heynen for the CA-p24 enzyme-linked immunosorbent assay and Berend Hooibrink (AMC Cell Biology) for live cell sorting. RNAi research in the Berkhout lab is sponsored by NWOCW (Top Grant) and ZonMw (Translational Gene Therapy Grant). E.H.-C received a postdoctoral fellowship from the MEC (Spanish Ministry of Education and Science, I-D+i 2008-2011).

References

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