This chapter contains sections titled: RNAi and HIV Therapeutics Transcriptional Gene Silencing Diversity of Viral Targets Gene Therapy Approaches for Treatment of HIV-1 Challenges to the Effective Use of RNAi in Anti-HIV Applications Conclusions References
To the Editors: We report successful treatment of disseminated Candida albicans infection in a child with a combination of antifungal agents and Mycograb (Neu Tec Pharma Plc, Manchester, U.K.), a human recombinant antibody against Candida heat shock protein 90 (hsp90). An 8-year-old girl with Down's syndrome and oral phobia underwent placement of a gastrostomy to assist with feeding. Her only significant past medical history was of a persistent ductus arteriosus which had been ligated in infancy. One week after insertion of the gastrostomy she presented to her local hospital with signs of peritonitis. A laparotomy the next day revealed a leaking gastrostomy, and multiple intra-abdominal abscesses were found. She underwent drainage of the abscesses, gastropexy and tightening of the gastrostomy, and therapy was commenced with intravenous cefuroxime and metronidazole. She was extubated postoperatively and sent to the ward where during the next 2 days she developed increasing respiratory distress, eventually requiring intubation and ventilation in the pediatric intensive care unit (PICU). She developed acute respiratory distress syndrome and had diffuse bilateral infiltrates and pleural effusions on chest radiograph. She developed septic shock requiring inotropic support. C. albicans and coagulase-negative staphylococci were subsequently isolated from peritoneal fluid taken at laparotomy, and the patient was treated with intravenous fluconazole and vancomycin on day 2 of admission to the PICU. She remained febrile and was difficult to ventilate and oxygenate with a persistent inotrope requirement. C. albicans was isolated in blood taken from an arterial catheter, central venous catheter and peripheral cultures, central venous catheter tip, arterial catheter tip, urine and 2 bronchoalveolar lavage specimens. She had 2 chest computerized tomography scans performed on days 11 and 24 of her PICU stay, both of which showed diffuse bilateral consolidation, atelectasis and effusions. A sonogram on days 13 and 19 of her PICU admission showed mitral valve papillary muscle vegetation; follow-up sonography on day 41 was normal. Inflammatory markers were abnormal intermittently. Various combinations of antifungal drugs were given: fluconazole (day 2–9) liposomal amphotericin B (day 9–18), flucytosine (day 17–24) and caspofungin (day 19–41). On day 20, Mycograb treatment was started for 7 days (1 mg/kg twice daily intravenously) because the patient was still clinically unstable and difficult to ventilate; this was well-tolerated with no apparent side effects. She was also given corticosteroids for inotrope-refractory shock at this stage. A short synacthen test was performed at this stage to check adrenal steroid responsiveness, the results of which were normal. On day 21, there was clinical improvement, and ventilation was weaned from high frequency oscillation to conventional ventilation. Between days 21 and 25 she continued to improve, with falling inotrope requirement. She received the last dose of Mycograb on day 26. On day 27, a bronchoalveolar lavage specimen was clear of C. albicans. The last positive blood culture for C. albicans was on day 13. She continued to improve clinically and was extubated to face mask ventilation on day 34. On day 45, she was discharged to the ward; on day 58, she was discharged home, fully recovered. This case is the first reported use of Mycograb in a child with Candida sepsis. In this case, C. albicans was isolated from several sites for several days. Her last positive culture for Candida was on day 13 of admission, but there was no clinical improvement in her systemic inflammatory response until day 21. She received several antifungal agents throughout her illness; therefore it is difficult to attribute her clinical improvement to any one antifungal agent, but progressive clinical improvement began 24 hours after adding Mycograb to caspofungin therapy. At the same time, corticosteroids were introduced for inotrope-refractory shock, however, she had a normal response to a short synacthen test, making the addition of steroids unlikely to have contributed significantly to her recovery. The last sonographic evidence of endocarditis was the day before Mycograb treatment was started. We did not observe any adverse effects as a result of the Mycograb infusions. In a double blind, placebo-controlled trial, Mycograb, an inhibitor of hsp90, given for 5 days in combination with liposomal amphotericin B resulted in an improved outcome compared with using liposomal amphotericin B alone in adults with invasive candidiasis.1 Hsp90 has recently been described as a “fungal Achilles' heel,” because the presence of an hsp90 inhibitor reduced fungal resistance to caspofungin or fluconazole in vitro.2 Although it is not possible to attribute the patient's recovery to any single agent, there was steady improvement clinically after the introduction of Mycograb. This is the first reported use of Mycograb in combination with caspofungin to treat a case of refractory candidiasis in a child. Helen Elizabeth Rowlands, MB, BS, MRCPCH Kevin Morris, MB, BS, MRCP, MD, FRCPCH Clive Graham, MB, BS, MRCP, MRCPath Birmingham Children's Hospital Birmingham, United Kingdom
RNA Interference (RNAi) effectors have been used to inhibit rogue RNAs in mammalian cells. However, rapidly evolving sequences such as the human immunodeficiency virus type 1 (HIV-1) require multiple targeting approaches to prevent the emergence of escape variants. Expressed long hairpin RNAs (lhRNAs) have recently been used as a strategy to produce multiple short interfering RNAs (siRNAs) targeted to highly variant sequences. We aimed to characterize the ability of expressed lhRNAs to generate independent siRNAs that silence three non-contiguous HIV-1 sites by designing lhRNAs comprising different combinations of siRNA-encoding sequences. All lhRNAs were capable of silencing individual target sequences. However, silencing efficiency together with concentrations of individual lhRNA-derived siRNAs diminished from the stem base (first position) towards the loop side of the hairpin. Silencing efficacy against HIV-1 was primarily mediated by siRNA sequences located at the base of the stem. Improvements could be made to first and second position siRNAs by adjusting spacing arrangements at their junction, but silencing of third position siRNAs remained largely ineffective. Although lhRNAs offer advantages for combinatorial RNAi, we show that good silencing efficacy across the span of the lhRNA duplex is difficult to achieve with sequences that encode more than two adjacent independent siRNAs.
Human immunodeficiency virus (HIV) is an attractive target for chimeric antigen receptor (CAR) therapy. CAR T cells have proved remarkably potent in targeted killing of cancer cells, and we surmised that CAR T cells could prove useful in eradicating HIV-infected cells. Toward this goal, we interrogate several neutralizing single-chain variable fragments (scFvs) that target different regions of the HIV envelope glycoprotein, gp120. We find here that CAR T cells with scFv from NIH45-46 antibody demonstrated the highest cytotoxicity. Although NIH45-46 CAR T cells are capable of eliminating antigen-expressing cells, we wanted to address HIV reactivation from ex vivo culture of HIV patient-derived CAR T cells. In order to capitalize on the HIV reactivation, we developed a conditionally replicating lentiviral vector (crLV). The crLV can hijack HIV machinery, forming a chimeric lentivirus (LV) instead of HIV and delivered to uninfected cells. We find that CAR T cells generated with crLVs have similar CAR-mediated functionality as traditional CARs. We also demonstrate crLVs' capability of expanding CAR percentage and protecting CD4 CAR T cell in HIV donors. Collectively, we demonstrate here that the novel crLV NIH45-46 CAR can serve as a strategy to combat HIV, as well as overcome HIV reactivation in CD4+ CAR T cells. Human immunodeficiency virus (HIV) is an attractive target for chimeric antigen receptor (CAR) therapy. CAR T cells have proved remarkably potent in targeted killing of cancer cells, and we surmised that CAR T cells could prove useful in eradicating HIV-infected cells. Toward this goal, we interrogate several neutralizing single-chain variable fragments (scFvs) that target different regions of the HIV envelope glycoprotein, gp120. We find here that CAR T cells with scFv from NIH45-46 antibody demonstrated the highest cytotoxicity. Although NIH45-46 CAR T cells are capable of eliminating antigen-expressing cells, we wanted to address HIV reactivation from ex vivo culture of HIV patient-derived CAR T cells. In order to capitalize on the HIV reactivation, we developed a conditionally replicating lentiviral vector (crLV). The crLV can hijack HIV machinery, forming a chimeric lentivirus (LV) instead of HIV and delivered to uninfected cells. We find that CAR T cells generated with crLVs have similar CAR-mediated functionality as traditional CARs. We also demonstrate crLVs' capability of expanding CAR percentage and protecting CD4 CAR T cell in HIV donors. Collectively, we demonstrate here that the novel crLV NIH45-46 CAR can serve as a strategy to combat HIV, as well as overcome HIV reactivation in CD4+ CAR T cells.
Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure. Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure.
Antimitotic drugs are key components of combination chemotherapy protocols for hematological and solid tumors. The taxanes (e.g., paclitaxel) bind to the β subunit of the tubulin heterodimer and reduce microtubule dynamics, leading to cell cycle arrest in G2/M. The effectiveness of combination chemotherapy is limited by tumor resistance to drugs initially or as a cumulative effect after several cycles of treatment. Because changes in the drug receptor may be linked to drug resistance, we investigated changes in β-tubulin isotypes in response to paclitaxel treatment in MCF7 breast cancer cells. We found that paclitaxel induced a 2-3 fold increase in mRNA for β-tubulin IIA and III genes, TUBB2A, and TUBB3. β-Tubulin class III protein increased; however, β-tubulin class II protein was not detected in these cells. Paclitaxel treatment following pretreatment with actinomycin D showed that the change in β-tubulin class III was due to increased transcription and linked to G2/M arrest. The increase in β-tubulin IIA mRNA was due to both enhanced stability and increased transcription, unassociated with G2/M arrest. We used micro-RNA superarrays to look for changes in families of micro-RNAs that might be linked to drug-induced changes in β-tubulin isotype mRNA and/or protein. We found a significant decrease in the tumor suppressor, miR-100, in MCF7 cells in response to paclitaxel treatment. Transfection of MCF7 cells with miR-100 significantly reduced β-tubulin I, IIA, IIB and V mRNA and prevented paclitaxel-induced increases in β-tubulin isotypes. This is the first report of a micro-RNA that regulates these specific β-tubulin isotype mRNAs.
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