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Retinitis pigmentosa, other inherited retinal diseases, and age-related macular degeneration lead to untreatable blindness because of the loss of photoreceptors. We have recently shown that transplantation of mouse photoreceptors can result in improved vision. It is therefore timely to develop protocols for efficient derivation of photoreceptors from human pluripotent stem (hPS) cells. Current methods for photoreceptor derivation from hPS cells require long periods of culture and are rather inefficient. Here, we report that formation of a transient self-organized neuroepithelium from human embryonic stem cells cultured together with extracellular matrix is sufficient to induce a rapid conversion into retinal progenitors in 5 days. These retinal progenitors have the ability to differentiate very efficiently into Crx(+) photoreceptor precursors after only 10 days and subsequently acquire rod photoreceptor identity within 4 weeks. Directed differentiation into photoreceptors using this protocol is also possible with human-induced pluripotent stem (hiPS) cells, facilitating the use of patient-specific hiPS cell lines for regenerative medicine and disease modeling. ispartof: STEM CELLS vol:31 issue:2 pages:408-414 ispartof: location:England status: published

lthough gammaretroviruses have been hugely im-portant for proof-of-principle demonstrations of efficacyin several specific clinical applications, other retroviral clas-ses, lentiviruses in particular, are emerging as promisingalternatives. Of the on-going 35 trials using lentivirus-basedvectors (LVs), 24 are in phase I, 9 in phase I/II, and 1 each inphases II and III (source: http://clinicaltrials.gov/). In ad-dition to their ability to stably integrate into the genome oftarget cells for long-term expression of the gene of interest,LVs enjoy a few significant advantages through their abilityto transduce nondividing cells and also to allow robusttransgene expression in cells where there is a high proba-bility of epigenetic silencing or position-related variation ofactivity (Trono, 2000; Schambach and Baum, 2008). Early-generation gammaretroviral vectors have been shown to beparticularly prone to insertional mutagenesis through inad-vertent long terminal repeat (LTR)-mediated upregulationof proto-oncogene expression (Ott et al., 2006; Hacein-Bey-Abina et al., 2008; Howe et al., 2008; Stein et al., 2010). Inpart, this risk may be enhanced by the intrinsic preference ofthese vectors to integrate in and around gene regulatoryregions. LVs may therefore have an overall safer integrationprofile, although both exhibit a semirandom pattern of in-sertion into the human genome, and this type of risk stillcannot be ignored (Bushman et al., 2005; Cavazzana-Calvoet al., 2010). The safety of LVs has been enhanced in severalways including self-inactivating (SIN) vector design, tat in-dependence, use of insulators, and employment of internaltissue- or lineage-specific promoter and enhancer sequences,thereby reducing risks of insertional mutagenesis and vectormobilization (Matrai et al., 2010). Even so, longer term ob-servations in the clinical arena will be needed to define therisk for newer integrating vectors with more clarity. Con-currently, the development of a stable packaging cell line toensure useful batch consistency and size during large-scaleclinical vector production and purification under strict GoodManufacturing Practice (GMP) conditions has been at-tempted by various groups. Inducible systems for generatingstable cell lines have been developed to eliminate cytotox-icity from viral gene products required for packaging, suchas the HIV-1-derived protease and Rev, and the heter-ologous VSV-G envelope protein (Xu et al., 2001; Ikeda et al.,2003; Cockrell et al., 2006; Broussau et al., 2008; Kutner et al.,2009; Throm et al., 2009). It is also noteworthy thatreplication-competent LVs (generated by homologous re-combination between packaging and vector constructs) havenot been detected in large-scale production batches based onlatest generation packaging systems and vector design(Merten et al., 2011). In this commentary, we comment on sixoriginal research articles addressing three separate areasrelating to continuing efforts in improving retroviral design,production, and application for preclinical research andclinical applications.In this issue of Human Gene Therapy, Marc Giry-Laterrie`reand colleagues describe a modified tetracycline (TET)-inducible, polycistronic vector system encoding a selectablemarker and allowing for simple transgene cloning. As notedin their paper, the individual features incorporated in the‘‘polyswitch vector’’ have been previously described byseveral groups. The novelty lies in the combination of thesein a single vector to facilitate stable, inducible, and reversibletransgene expression from a single integration event. Sincethe description of the first tetracycline-inducible system byGossen and Bujard (1992), various small-molecule inducersystems have been employed to control the levels and timingof transgene expression both in vitro and in vivo (Vigna et al.,2002; Sirin and Park, 2003; Galimi et al., 2005). However,many have suffered from either low levels of inducibility orleakiness in the absence of the specific inducer. The modifiedpTF promoter used in this current study was shown to havelow basal activity and high inducibility. The authors alsoseparated a selectable marker (regulated by a ubiquitoushuman elongation factor-1a promoter) from the inducibletransgene cassette for enrichment of transduced cells beforeactivating the transgene of interest, and were able to dem-onstrate regulated expression from their vector in cell lines aswell as primary human cells. Of course, it will now beimportant to evaluate the performance of this vector inpreclinical animal models, especially in terms of drug in-ducibility and reversibility on withdrawal. Although this islikely to be useful for a number of applications, there willalso be restrictions on the nature of the transgene dictated

Gene therapy presents an attractive alternative to allogeneic haematopoietic stem cell transplantation (HSCT) for treating patients suffering from primary immunodeficiency disorder (PID). The conceptual advantage of gene correcting a patient's autologous HSCs lies in minimizing or completely avoiding immunological complications arising from allogeneic transplantation while conferring the same benefits of immune reconstitution upon long-term engraftment. Clinical trials targeting X-linked chronic granulomatous disorder (X-CGD) have shown promising results in this context. However, long-term clinical benefits in these patients have been limited by issues of poor engraftment of gene-transduced cells coupled with transgene silencing and vector induced clonal proliferation. Novel vectors incorporating safety features such as self-inactivating (SIN) mutations in the long terminal repeats (LTRs) along with synthetic promoters driving lineage-restricted sustainable expression of the gp91phox transgene are expected to resolve the current pitfalls and require rigorous preclinical testing. In this chapter, we have outlined a protocol in which X-CGD mouse model derived induced pluripotent stem cells (iPSCs) have been utilized to develop a platform for investigating the efficacy and safety profiles of novel vectors prior to clinical evaluation.

Lentiviral vectors (lentivectors) are effective for stimulation of cell-mediated and humoral immunity following subcutaneous and intramuscular immunization. However, lentivector genome integration carries a risk of perturbation of host gene expression. Here, we demonstrate that lentivectors with multiple mutations that prevent integration are also effective immunogens. First, systemic CD8 + T-cell responses to the model antigen ovalbumin were detected following subcutaneous injection of nonintegrating lentivectors. Transfer of transgenic OT1 T cells demonstrated that antigen presentation persisted for at least 30 days. Furthermore, an enhanced CD8 + T-cell response, peaking at 7 days, was stimulated by coexpression of p38 MAP kinase or an NF-κB activator from the same vector. Second, we demonstrated systemic CD8 + T-cell and antibody responses to the secreted hepatitis B virus (HBV) surface antigen expressed from a nonintegrating lentivector injected intramuscularly. The induction, specificity, and kinetics of antibody production closely mimicked those of natural HBV infection. In this case, both the vector genome and the immune response were maintained for at least 2 months. Together, our data indicate that nonintegrating lentivectors can be employed to generate effective vaccines.

Human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS in humans, exhibits a very high rate of recombination. Bearing in mind the significant epidemiological and clinical contrast between HIV-2 and HIV-1 as well as the critical role that recombination plays in viral evolution, we examined the nature of HIV-2 recombination. Towards this end, a strategy was devised to measure the rate of crossover of HIV-2 by evaluating recombinant progeny produced exclusively by heterodimeric virions. The results showed that HIV-2 exhibits a crossover rate similar to that of HIV-1 and murine leukemia virus, indicating that the extremely high rate of crossover is a common retroviral feature.

The employment of HIV-1-based vectors in clinical trials is controversial mainly due to the lethal nature of the virus. HIV-2 is less pathogenic in nature and therefore is likely to be safer for vector design and production. We developed HIV-2-based self-inactivating vectors in which 520 bp out of 554 bp of the viral U3 was deleted. Interestingly, high titers were obtained only when an exogenous promoter was used to drive expression of viral RNA. It was found that the vectors could target a wide range of mammalian cell types including primary neuronal cells and could yield long term expression. It is also noteworthy that the HIV-2 vectors could be effectively cross-packaged into HIV-1 core, which might provide for enhanced safety by reducing the recombination potential of the system.

Murine models of human genetic disorders provide a valuable tool for investigating the scope for application of induced pluripotent stem cells (iPSC). Here we present a proof-of-concept study to demonstrate generation of iPSC from a mouse model of X-linked chronic granulomatous disease (X-CGD), and their successful differentiation into haematopoietic progenitors of the myeloid lineage. We further demonstrate that additive gene transfer using lentiviral vectors encoding gp91(phox) is capable of restoring NADPH-oxidase activity in mature neutrophils derived from X-CGD iPSC. In the longer term, correction of iPSC from human patients with CGD has therapeutic potential not only through generation of transplantable haematopoietic stem cells, but also through production of large numbers of autologous functional neutrophils.