Human Gene Therapy

PhD-trained biomedical scientists are moving into an increasingly diverse variety of careers within the sciences. However, graduate and postdoctoral training programs have historically focused on academic career preparation, and have not sufficiently prepared trainees for transitioning into other scientific careers. Advocates for science have raised the concern that the collective disregard of the broader career-development needs for predoctoral and postdoctoral trainees could drive talent away from science in upcoming generations. A shift is occurring, wherein universities are increasingly investing in centralized career development programs to address this need. In this Perspective, I reflect on the movement that brought biomedical PhD career development to the spotlight in recent years, and how this movement has influenced both the academic biomedical community and the field of career development. I offer recommendations for universities looking to establish or strengthen their career development programs, including recommendations for how to develop a campus culture that values career development as part of pre- and postdoctoral training. I also suggest steps that faculty might take to facilitate the career development of their mentees, regardless of the mentee's career aspirations. Finally, I reflect on recent national efforts to incentivize innovation, evaluation, and research in the field of biomedical PhD career development, and propose actions that the scientific community can take to support biomedical career development further as a scholarly discipline. These investments will enable new approaches to be rigorously tested and efficiently disseminated to support this rapidly growing field. Ultimately, strengthening biomedical career development will be essential for attracting the best talent to science and helping them efficiently move into careers that will sustain our nation's scientific enterprise.

Liver fibrosis results in cirrhosis, liver cancer, and liver failure, which is a major cause of mortality worldwide. Gene therapy is a relatively new paradigm in medicine, with enormous therapeutic potential. The development of an efficient and safe delivery system is essential for clinical gene therapy. In the present study, we evaluated augmenter of liver regeneration/growth factor ERV1-like (ALR/GFER) gene therapeutic effect mediated by a novel minicircle vector (MC-hALR). The results in liver fibrotic rats that received MC-hALR through hydrodynamics-based transfection (HBT) for 8 weeks indicated that the minicircle DNA vector produced a more effective gene therapy effect than traditional plasmids (pcDNA3.1-hALR). Even when we reduced the treatment dose of MC-hALR to 30% (w/w) and the treatment frequency from weekly to biweekly, the in vitro and in vivo results still demonstrated that higher ALR gene expression significantly blocked increases in transforming growth factor-β1 (TGF-β1), platelet derived growth factor-BB (PDGF-BB), and α-smooth muscle aorta (α-SMA) levels; effectively suppressed the production of collagens, especially collagen I; and effectively alleviated liver injury and fibrosis in rats, thereby improving the survival rate of liver fibrotic rats. It is preliminarily concluded that the relative overexpression of MC-hALR inhibits the activation of hepatic stellate cells (HSCs), thereby alleviating liver fibrosis in rats.

Adeno-associated virus (AAV) gene transfer is a promising treatment for genetic abnormalities. Optimal AAV vectors are showing success in clinical trials. Gene transfer to skeletal muscle and liver is being explored as a potential therapy for some conditions, that is, α₁-antitrypsin (AAT) disorder and hemophilia B. Exploring approaches that enhance transduction of liver and skeletal muscle, using these vectors, is beneficial for gene therapy. Regulating hormones as an approach to improve AAV transduction is largely unexplored. In this study we tested whether insulin therapy improves liver and skeletal muscle gene transfer. In vitro studies demonstrated that the temporary coadministration (2, 8, and 24 hr) of insulin significantly improves AAV2-CMV-LacZ transduction of cultured liver cells and differentiated myofibers, but not of lung cells. In addition, there was a dose response related to this improved transduction. Interestingly, when insulin was not coadministered with the virus but given 24 hr afterward, there was no increase in the transgene product. Insulin receptor gene (INSR) expression levels were increased 5- to 13-fold in cultured liver cells and differentiated myofibers when compared with lung cells. Similar INSR gene expression profiles occurred in mouse tissues. Insulin therapy was performed in mice, using a subcutaneously implanted insulin pellet or a high-carbohydrate diet. Insulin treatment began just before intramuscular delivery of AAV1-CMV-schFIX or liver-directed delivery of AAV8-CMV-schFIX and continued for 28 days. Both insulin augmentation therapies improved skeletal muscle- and liver-directed gene transduction in mice as seen by a 3.0- to 4.5-fold increase in human factor IX (hFIX) levels. The improvement was observed even after the insulin therapy ended. Monitoring insulin showed that insulin levels increased during the brief period of rAAV delivery and during the entire insulin augmentation period (28 days). This study demonstrates that AAV transduction of liver or skeletal muscle can be improved by insulin therapy.

Mucopolysaccharidosis type II (MPS II) is a rare X-linked genetic disorder caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS), leading to impaired catabolism of ubiquitous polysaccharides and abnormal accumulation of these undegraded substrates in the lysosome. Like many lysosomal storage diseases, MPS II is characterized by both somatic and central nervous system (CNS) involvement. Intravenous enzyme replacement therapy can improve somatic manifestations of MPS II, but systemic IDS does not cross the blood–brain barrier and therefore cannot address CNS disease. In this study, an adeno-associated virus serotype 9 vector carrying the IDS gene was injected into the cerebrospinal fluid (CSF) of IDS deficient mice, a model of MPS II. Treated mice exhibited dose-dependent IDS expression and resolution of brain storage lesions, as well as improvement in long-term memory in a novel object recognition test. These findings suggest that delivery of adeno-associated virus vectors into CSF could serve as a platform for efficient, long-term enzyme delivery to the CNS, potentially addressing this critical unmet need for patients with MPS II and many related lysosomal enzyme deficiencies.

Alipogene tiparvovec (Glybera) is a gene therapy product approved in Europe under the “exceptional circumstances” pathway as a treatment for lipoprotein lipase deficiency (LPLD), a rare genetic disease resulting in chylomicronemia and a concomitantly increased risk of acute and recurrent pancreatitis, with potentially lethal outcome. This retrospective study analyzed the frequency and severity of pancreatitis in 19 patients with LPLD up to 6 years after a single treatment with alipogene tiparvovec. An independent adjudication board of three pancreas experts, blinded to patient identification and to pre- or post-gene therapy period, performed a retrospective review of data extracted from the patients' medical records and categorized LPLD-related acute abdominal pain events requiring hospital visits and/or hospitalizations based on the adapted 2012 Atlanta diagnostic criteria for pancreatitis. Both entire disease time period data and data from an equal time period before and after gene therapy were analyzed. Events with available medical record information meeting the Atlanta diagnostic criteria were categorized as definite pancreatitis; events treated as pancreatitis but with variable levels of laboratory and imaging data were categorized as probable pancreatitis or acute abdominal pain events. A reduction of approximately 50% was observed in all three categories of the adjudicated post-gene therapy events. Notably, no severe pancreatitis and only one intensive care unit admission was observed in the post-alipogene tiparvovec period. However, important inter- and intraindividual variations in the pre- and post-gene therapy incidence of events were observed. There was no relationship between the posttreatment incidence of events and the number of LPL gene copies injected, the administration of immunosuppressive regimen or the percent triglyceride decrease achieved at 12 weeks (primary end point in the prospective clinical studies). Although a causal relationship cannot be established and despite the limited number of individuals evaluated, results from this long-term analysis suggest that alipogene tiparvovec was associated with a lower frequency and severity of pancreatitis events, and a consequent overall reduction in health care resource use up to 6 years posttreatment.

Immunomodulation has been regarded as an important therapeutic aspect of mesenchymal stromal cell–derived extracellular vesicles (MSC-EVs) in renal ischemic reperfusion injury (IRI), and the specific mechanism still unclear. Here, we attempt to test the function of human MSC-EVs on renal IRI by targeting the natural killer (NK) cells and to investigate the possible mechanism. Data indicated that EVs decreased NK cells in spleen and ischemic kidney. Both the EVs and antibody-dependent depletion of NK cells displayed a protective role in IRI rats. Moreover, the splenectomy model was established to evaluate the role of spleen in this process. It showed that the NK cell regulatory ability and renal protective effects by EVs still exist without spleen, which is unlike MSC properties published previously. Further, the down-regulation of chemokines in injured kidney and the delivery of RNAs through EVs in vitro were also observed. Through the microRNA array test, various inflammation-related microRNAs highly expressed in MSC-EVs compared with fibroblast EVs were tested. Thus, these results indicated that MSC-EVs could ameliorate renal ischemic reperfusion injury by decreasing NK cells and the spleen is not necessary in this process. The regulation of chemokines in injured kidney was the other factor, and the transfer of various microRNAs in the MSC-EVs may be involved. This provides direction for future clinical applications.

Gene therapy techniques aiming to induce the long-term interferon-β (IFN-β) expression are desirable for inhibiting cancer growth. However, there has been no success in this regard because IFN-β significantly inhibits transgene expression. This study used the IFN-inducible Mx promoter to promote IFN-β expression. The pMx-IFN-β plasmid was constructed to achieve long-term IFN-β expression. In cultured cells transfected with the Mx promoter-driven reporter protein plasmid, IFN-β induced concentration-dependent expression of the reporter protein. After the hydrodynamic injection of pMx-IFN-β into mice, the serum concentration of IFN-β was maintained at ≥100 pg/mL for >1 month. IFN-β expression was significantly suppressed by the co-injection of small interfering RNA targeting the interferon-α/β receptor (IFNAR), suggesting that IFN-β binding to IFNAR increased IFN-β expression. Moreover, the hydrodynamic injection of pMx-IFN-β significantly suppressed the growth of colon26 tumors in mice. In contrast, a conventional promoter-driven plasmid was less effective than pMx-IFN-β in all the experiments. Taken together, these results indicate that the interferon-inducible Mx promoter-driven expression system effectively achieves long-term expression of IFN-β and represents a potential tool for cancer gene therapy.