Development of Hematopoietic Stem Cell Based Gene Therapy for HIV-1 Infection: Considerations for Proof of Concept Studies and Translation to Standard Medical Practice

Over the past 15 years we have been investigating an alternative approach to treating HIV-1/AIDS, based on the creation of a disease-resistant immune system through transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and progenitor cells (GM-HSPC). We propose that the expression of selected RNA-based HIV-1 inhibitors in the CD4+ cells derived from GM-HSPC will protect them from HIV-1 infection and results in a sufficient immune repertoire to control HIV-1 viremia resulting in a functional cure for HIV-1/AIDS. Additionally, it is possible that the subset of protected T cells will also be able to facilitate the immune-based elimination of latently infected cells if they can be activated to express viral antigens. Thus, a single dose of disease resistant GM-HSPC could provide an effective treatment for HIV-1+ patients who require (or desire) an alternative to lifelong antiretroviral chemotherapy. We describe herein the results from several pilot clinical studies in HIV-1 patients and our strategies to develop second generation vectors and clinical strategies for HIV-1+ patients with malignancy who require ablative chemotherapy as part of treatment and others without malignancy. The important issues related to stem cell source, patient selection, conditioning regimen and post-infusion correlative studies become increasingly complex and are discussed herein.     

Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy

In HIV-1-infected individuals on currently recommended antiretroviral therapy (ART), viremia is reduced to <50 copies of HIV-1 RNA per milliliter, but low-level residual viremia appears to persist over the lifetimes of most infected individuals. There is controversy over whether the residual viremia results from ongoing cycles of viral replication. To address this question, we conducted 2 prospective studies to assess the effect of ART intensification with an additional potent drug on residual viremia in 9 HIV-1-infected individuals on successful ART. By using an HIV-1 RNA assay with single-copy sensitivity, we found that levels of viremia were not reduced by ART intensification with any of 3 different antiretroviral drugs (efavirenz, lopinavir/ritonavir, or atazanavir/ritonavir). The lack of response was not associated with the presence of drug-resistant virus or suboptimal drug concentrations. Our results suggest that residual viremia is not the product of ongoing, complete cycles of viral replication, but rather of virus output from stable reservoirs of infection.     

A Novel High Throughput,Parallel Infection Assay for Determining the Replication Capacities of 346 Primary HIV-1 Isolates of the Zurich Primary HIV-1 Infection Study in Primary Cells

HIV-1 replication capacity is an important characteristic to understand the replication competence of single variants or virus populations. It can further aid in the understanding of HIV-1 pathogenicity, disease progression, and drug resistance mutations. To effectively study RC, many assays have been established. However, there is still demand for a high throughput replication capacity assay using primary cells which is robust and reproducible. In this study, we established such an assay and validated it using 346 primary HIV-1 isolates from patients enrolled in the Zurich Primary HIV Infection study (ZPHI) and two control viruses, HIV-1 JR-CSF_WT and HIV-1 JR-CSF_{K65R_M184V}. Replication capacity was determined by measuring the viral growth on PBMCs over 10 days by longitudinally transferring cell culture supernatant to TZM-bl reporter cells. By utilizing the TZM-bl luciferase reporter assay, we determined replication capacity by measuring viral infectivity. The simplicity of the experimental setup allowed for all 346 primary HIV-1 isolates to be replicated at one time. Although the infectious input dose for each virus was normalized, a broad range of replication capacity values over 4 logs was observed. The approach was confirmed by two repeated experiments and we demonstrated that the reproducibility of the replication capacity values is statistically comparable between the two separate experiments. In summary, these results endorse our high throughput replication capacity assay as reproducible and robust and can be utilized for large scale HIV-1 replication capacity experiments in primary cells.     

Nanomedicine based approaches for the delivery of siRNA in cancer

Small interfering RNA (siRNA) technology holds great promise as a therapeutic intervention for targeted gene silencing in cancer and other diseases. However, in vivo systemic delivery of siRNA‐based therapeutics to tumour tissues/cells remains a challenge. The major limitations against the use of siRNA as a therapeutic tool are its degradation by serum nucleases, poor cellular uptake and rapid renal clearance following systemic administration. Several siRNA‐based loco‐regional therapeutics are already in clinical trials. Further development of siRNAs for anti‐cancer therapy depends on the development of safe and effective nanocarriers for systemic administration. To overcome these hurdles, nuclease‐resistant chemically modified siRNAs and variety of synthetic and natural biodegradable lipids and polymers have been developed to systemically deliver siRNA with different efficacy and safety profiles. Cationic liposomes have emerged as one of the most attractive carriers because of their ability to form complexes with negatively charged siRNA and high in vitro transfection efficiency. However, their effectiveness as potential therapeutic carriers is limited by potential for pulmonary toxicity. Recently, our laboratories described the use of neutral 1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylcholine based nanoliposomes in murine tumour models. We found this approach to be safe and 10‐ and 30‐fold more effective than cationic liposomes and naked siRNA, respectively, for systemic delivery of siRNA into tumour tissues. Here, we review potential approaches for systemic delivery of siRNA for cancer therapy.     

HIV-Ⅰ gag基因在大肠杆菌中的表达

目的表达HIV-1型鹳基因蛋白。方法将编码HIV-1型gag的部分序列,克隆到表达载体pET-28a后,进行IPTG诱导在E．coli BL21（DE3）表达,表达产物分别经SDS-PAGE和Western-Blot进行检测。结果表达产物为分子量约50kD的融合蛋白,并可与HIV-1型病人阳性血清发生特异性反应。结论鹳基因在大肠杆菌中得到表达,且表达产物具有良好的抗原性。     

Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection

The precise identification of the HIV-1 envelope glycoprotein (Env) responsible for productive clinical infection could be instrumental in elucidating the molecular basis of HIV-1 transmission and in designing effective vaccines. Here, we developed a mathematical model of random viral evolution and, together with phylogenetic tree construction, used it to analyze 3,449 complete env sequences derived by single genome amplification from 102 subjects with acute HIV-1 (clade B) infection. Viral env genes evolving from individual transmitted or founder viruses generally exhibited a Poisson distribution of mutations and star-like phylogeny, which coalesced to an inferred consensus sequence at or near the estimated time of virus transmission. Overall, 78 of 102 subjects had evidence of productive clinical infection by a single virus, and 24 others had evidence of productive clinical infection by a minimum of two to five viruses. Phenotypic analysis of transmitted or early founder Envs revealed a consistent pattern of CCR5 dependence, masking of coreceptor binding regions, and equivalent or modestly enhanced resistance to the fusion inhibitor T1249 and broadly neutralizing antibodies compared with Envs from chronically infected subjects. Low multiplicity infection and limited viral evolution preceding peak viremia suggest a finite window of potential vulnerability of HIV-1 to vaccine-elicited immune responses, although phenotypic properties of transmitted Envs pose a formidable defense.     

Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment

RNA interference (RNAi) represents a promising strategy for identification and validation of putative therapeutic targets and for treatment of a myriad of important human diseases including cancer. However, the effective systemic in vivo delivery of small interfering RNA (siRNA) to tumors remains a formidable challenge. Using a robust self-assembly strategy, we develop a unique nanoparticle (NP) platform composed of a solid polymer/cationic lipid hybrid core and a lipid-poly(ethylene glycol) (lipid-PEG) shell for systemic siRNA delivery. The new generation lipid–polymer hybrid NPs are small and uniform, and can efficiently encapsulate siRNA and control its sustained release. They exhibit long blood circulation (t_1/2 ∼8 h), high tumor accumulation, effective gene silencing, and negligible in vivo side effects. With this RNAi NP, we delineate and validate the therapeutic role of Prohibitin1 (PHB1), a target protein that has not been systemically evaluated in vivo due to the lack of specific and effective inhibitors, in treating non-small cell lung cancer (NSCLC) as evidenced by the drastic inhibition of tumor growth upon PHB1 silencing. Human tissue microarray analysis also reveals that high PHB1 tumor expression is associated with poorer overall survival in patients with NSCLC, further suggesting PHB1 as a therapeutic target. We expect this long-circulating RNAi NP platform to be of high interest for validating potential cancer targets in vivo and for the development of new cancer therapies.With the capability to silence any gene of interest, RNA interference (RNAi) technology has demonstrated enormous potential in medical research and applications (1, 2). RNAi-mediated gene silencing has revealed the functionality of specific genetic alterations in cancers (3–5). Many of these genes and pathways are considered “undruggable” targets or require complex and time-consuming development of effective inhibitors. The ubiquitous application of RNAi in cancer research and therapy is nevertheless hindered by the challenge of effective systemic in vivo delivery of siRNA to tumors, which requires overcoming of multiple physiological barriers, such as enzymatic degradation, rapid elimination by renal excretion or by the mononuclear phagocyte system (MPS), and poor cellular uptake and endosomal escape (2, 6, 7). To this end, a great number of cationic lipid/polymer-based nanoparticles (NPs) have been developed to protect siRNA from serum nucleases and facilitate its cytosolic delivery (8). Surface PEGylation has also been applied extensively to improve NP stability and reduce MPS recognition (9, 10). Several RNAi nanotherapeutics are now in clinical trial in cancer patients. However, the clinical stage anticancer RNAi NPs have shown relatively rapid clearance in blood (11, 12), which may reduce their extravasation into tumor tissue through the enhanced permeability and retention (EPR) effect (13). This could result in decreased in vivo silencing efficacy and consequently limit potential clinical impact.Here we report the development of a long-circulating RNAi NP platform for exploring and validating PHB1 as a potential new therapeutic target in NSCLC treatment. This lipid–polymer hybrid small interfering RNA (siRNA) NP (Fig. 1A) is rationally developed through a robust self-assembly approach, and has a unique nanostructure comprising a cationic lipid/siRNA complex-containing poly(D,l-lactide-co-glycolide) (PLGA) polymer core and a lecithin/lipid-PEG shell. Unlike previous lipid–polymer hybrid RNAi NPs formulated by the double emulsion and solvent evaporation techniques (14–18), our self-assembled hybrid NP is much smaller (≤100 nm) and exhibits promising in vivo features for systemic siRNA delivery, including long circulation, high tumor accumulation, effective gene silencing, and modest side effects. We also reveal that the cationic lipid component and the surface lipid-PEG have a critical role in controlling gene silencing efficacy and pharmacokinetics (PK) of the new generation hybrid NPs. After systematic investigation and screening, this siRNA NP is successfully applied to define the role of PHB1 as a potential cancer target. Although proposed to modulate tumor cell proliferation and chemoresistance (19–21), systemic in vivo validation of PHB1 for cancer treatment remains elusive. In this work, we demonstrate antitumor activity following systemic delivery of PHB1 siRNA and propose this approach as a novel therapeutic modality for NSCLC treatment.Open in a separate windowFig. 1.Self-assembly of lipid–polymer hybrid NP for siRNA delivery. (A) Schematic diagram of the NP structure and the self-assembly process for NP formulation. (B) Monitoring of the NP formulation by dynamic light scattering (DLS). No particle formation was observed when H₂O was added to the acetone solution of PLGA/G0-C14 in a volume ratio of 1:20. When aqueous siRNA was added to the same solution, small complexes can be detected with a size of ∼26 nm. After nanoprecipitation in the bulk aqueous solution of lecithin/lipid-PEG, the hybrid NPs show a size of ∼100 nm. Inset is the transmission electron microscopy (TEM) image of the NPs. (C) Luciferase expression in Luc-HeLa cells transfected with NP(siLuc) composed of different cationic lipids or lipid-like compounds. All siRNA NPs were made with the N/P ratio of 10/1. Lipo2K was used as a positive control. (D) Sustained luciferase silencing by NP(siLuc) vs. Lipo2K–siLuc complexes. (E) NP uptake in the presence of specific endocytic inhibitors.     

Intraneuronal β-Amyloid Accumulation: Aging HIV-1 Human and HIV-1 Transgenic Rat Brain

The prevalence of HIV-1 associated neurocognitive disorders (HAND) is significantly greater in older, relative to younger, HIV-1 seropositive individuals; the neural pathogenesis of HAND in older HIV-1 seropositive individuals, however, remains elusive. To address this knowledge gap, abnormal protein aggregates (i.e., β-amyloid) were investigated in the brains of aging (>12 months of age) HIV-1 transgenic (Tg) rats. In aging HIV-1 Tg rats, double immunohistochemistry staining revealed abnormal intraneuronal β-amyloid accumulation in the prefrontal cortex (PFC) and hippocampus, relative to F344/N control rats. Notably, in HIV-1 Tg animals, increased β-amyloid accumulation occurred in the absence of any genotypic changes in amyloid precursor protein (APP). Furthermore, no clear amyloid plaque deposition was observed in HIV-1 Tg animals. Critically, β-amyloid was co-localized with neurons in the cortex and hippocampus, supporting a potential mechanism underlying synaptic dysfunction in the HIV-1 Tg rat. Consistent with these neuropathological findings, HIV-1 Tg rats exhibited prominent alterations in the progression of temporal processing relative to control animals; temporal processing relies, at least in part, on the integrity of the PFC and hippocampus. In addition, in post-mortem HIV-1 seropositive individuals with HAND, intraneuronal β-amyloid accumulation was observed in the dorsolateral PFC and hippocampal dentate gyrus. Consistent with observations in the HIV-1 Tg rat, no amyloid plaques were found in these post-mortem HIV-1 seropositive individuals with HAND. Collectively, intraneuronal β-amyloid aggregation observed in the PFC and hippocampus of HIV-1 Tg rats supports a potential factor underlying HIV-1 associated synaptodendritic damage. Further, the HIV-1 Tg rat provides a biological system to model HAND in older HIV-1 seropositive individuals.     

深圳市HIV-1分子流行病学及不同亚型混合感染研究

目的 了解深圳市HIV分子流行病学及不同亚型混合感染情况.方法 对429例HIV-1感染者进行详细的流行病学调查,提取外周静脉抗凝血中的病毒核酸,RT-PCR巢式扩增病毒的包膜蛋白(env)和核心蛋白(gog)基因的核酸片段,结合HIV数据库和Mega软件分析测序结果,建立进化树,确定每个基因的亚型.结果 在400份合...     

套式PCR对HIV—l env基因检测的敏感度分析

采用套式PCR对含HIV—1膜蛋白基因的B亚型质粒和HIV—1感染者外周血单核细胞(PBMC)进行env基因部分片断的扩增,套式PCR可使常规PCR的敏感性提高100至1000倍,常规PCR只扩增出12份PBMC中的2份样品,套式PCR则扩增出全部样品中HIV—1膜蛋白基因片断.     

Specificity of the HIV-1 Protease on Substrates Representing the Cleavage Site in the Proximal Zinc-Finger of HIV-1 Nucleocapsid Protein

To explore the sequence context-dependent nature of the human immunodeficiency virus type 1 (HIV-1) protease’s specificity and to provide a rationale for viral mutagenesis to study the potential role of the nucleocapsid (NC) processing in HIV-1 replication, synthetic oligopeptide substrates representing the wild-type and modified versions of the proximal cleavage site of HIV-1 NC were assayed as substrates of the HIV-1 protease (PR). The S1′ substrate binding site of HIV-1 PR was studied by an in vitro assay using KIVKCF↓NCGK decapeptides having amino acid substitutions of N17 residue of the cleavage site of the first zinc-finger domain, and in silico calculations were also performed to investigate amino acid preferences of S1′ site. Second site substitutions have also been designed to produce “revertant” substrates and convert a non-hydrolysable sequence (having glycine in place of N17) to a substrate. The specificity constants obtained for peptides containing non-charged P1′ substitutions correlated well with the residue volume, while the correlation with the calculated interaction energies showed the importance of hydrophobicity: interaction energies with polar residues were related to substantially lower specificity constants. Cleavable “revertants” showed one residue shift of cleavage position due to an alternative productive binding mode, and surprisingly, a double cleavage of a substrate was also observed. The results revealed the importance of alternative binding possibilities of substrates into the HIV-1 PR. The introduction of the “revertant” mutations into infectious virus clones may provide further insights into the potential role of NC processing in the early phase of the viral life-cycle.     

Evaluation of the use of dried spots and of different storage conditions of plasma for HIV-1 RNA quantification

OBJECTIVES: The aim of the study was to evaluate the use of dried plasma spots to determine HIV-1 RNA viral loads. METHODS: The viral loads of 30 liquid plasma samples were compared with those of corresponding dried plasma spots on filter paper (DPS-FP) and in tubes (DPS-T), both of which were left for 7 days at 22 degrees C. Also, 10 liquid plasma samples with detectable viral load were stored at 4, 22 or 37 degrees C for 7 days and five further liquid plasma samples were air-dried for up to 54 h to assess the effects of temperature and the drying step on HIV-1 viral load. RESULTS: The viral loads of the 30 liquid plasma samples correlated significantly with those of the paired dried spots DPS-FP and DPS-T, but with median losses of 0.64 and 0.69 log(10) HIV-1 RNA copies/mL, respectively, and a limit of detection of 3 log(10) copies/mL. The 10 liquid plasma samples stored for 1 week at 37 degrees C showed a weaker correlation and had a significantly reduced median viral load (-0.92 log(10); P=0.005) when compared with the viral load of the matched plasma stored at - 80 degrees C. Most of the loss happened during the drying step. CONCLUSIONS: Reliable measurement of HIV-1 RNA viral load requires good plasma storage conditions. HIV RNA stability was affected by desiccation and 1 week of storage at 37 degrees C. However, our findings suggest that liquid plasma can be kept at 4 or 22 degrees C for a week with no effect on viral load.     

Evaluation of the HIV-1 Polymerase Gene Sequence Diversity for Prediction of Recent HIV-1 Infections Using Shannon Entropy Analysis

HIV-1 incidence is an important parameter for assessing the impact of HIV-1 interventions. The aim of this study was to evaluate HIV-1 polymerase (pol) gene sequence diversity for the prediction of recent HIV-1 infections. Complete pol Sanger sequences obtained from 45 participants confirmed to have recent or chronic HIV-1 infection were used. Shannon entropy was calculated for amino acid (aa) sequences for the entire pol and for sliding windows consisting of 50 aa each. Entropy scores for the complete HIV-1 pol were significantly higher in chronic compared to recent HIV-1 infections (p < 0.0001) and the same pattern was observed for some sliding windows (p-values ranging from 0.011 to <0.001), leading to the identification of some aa mutations that could discriminate between recent and chronic infection. Different aa mutation groups were assessed for predicting recent infection and their performance ranged from 64.3% to 100% but had a high false recency rate (FRR), which was decreased to 19.4% when another amino acid mutation (M456) was included in the analysis. The pol-based molecular method identified in this study would not be ideal for use on its own due to high FRR; however, this method could be considered for complementing existing serological assays to further reduce FRR.