Gene transfer technologies and new gene therapy strategies

SR-Tiget Unit - ERC Advanced Grant
Luigi Naldini, Head of Unit

Research activities (updated 2013)

 1. Improving hematopoietic stem cell gene therapy and regulated vectors

Bernhard Gentner (Project Leader), Erika Zonari (post-doc), Francesco Boccalatte (Ph.D. student), Tiziana Plati (Lab technician), Lucia Sergi Sergi (Lab Technician)

Hematopoietic stem cell (HSC) gene therapy has made significant progress over the last years and currently offers a therapeutic option for several genetic diseases, potentially less risky and more effective than allogeneic stem cell transplantation. Recent clinical trials performed at TIGET have demonstrated that ex vivo transduction with lentiviral vectors can result in near-complete and stable gene marking of hematopoiesis in vivo, compatible with successful genetic modification and maintenance of long-term repopulating HSC. This project is aimed at further increasing the efficacy and safety of HSC gene therapy in order to ensure sustainability and broaden its application to a wide range of diseases.

1.1 Improving purity and ex vivo culture of HSC

The need for ex vivo culture and cytokine stimulation of HSC remains a potential concern of current transduction protocols, since these interventions might reduce HSC and progenitor cell fitness as compared to unmanipulated cells, resulting in a longer duration of aplasia and the need to infuse a higher number of cells which might become a limiting factor especially in adult patients. Recently, several protocols have been published which might allow a better maintenance or even expansion of HSC in ex vivo culture. Unfortunately, results are variable and not always reproducible, and an in-depth understanding of the biological mechanism of HSC maintenance as well as their surface markers in culture is lacking. We aim to better preserve long-term engrafting cells in ex vivo culture, identify surface- and molecular markers to prospectively identify HSC during prolonged culture and set up high throughput surrogate assays which allow modulating culture conditions towards improved HSC maintenance. We are testing published and novel compounds/interventions for their capacity to maintain HSC in culture, including antagonism of specific miRNAs that we have found to be involved in the regulation of HSC cell cycle and differentiation (see below). The possibility to expand HSC in culture would overcome a major bottleneck in HSC gene therapy and allow implementing novel gene therapy technologies such as gene correction by targeted integration (see below). A major shortcoming of HSC ex vivo cultures is the lack of physical separation of cells at different maturation stages provided by the physiologic environment of the bone marrow niche. Indeed, differentiated cells release paracrine factors jeopardizing HSC maintenance. Thus, the higher the HSC purity in the culture, the better HSC will be maintained. Currently, the standard approach for HSC purification is positive selection for the surface marker CD34. We are investigating novel purification approaches allowing to increase HSC purity and scale down the culture volume. This will give significant advantages in terms of HSC maintenance, but also in terms of vector requirement for clinical use, a limiting factor especially when treating adult patients and more prevalent diseases, as in the case of gene therapy for thalassemia.

1.2 Designing vectors with tigthly regulated transgene expression

Long term gene modification/correction of hematopoiesis requires integration of the transgene cassette into HSC. However, in most cases, transgene expression is not required in HSC in order to achieve therapeutic benefit. On the contrary, ectopic/unregulated transgene expression in HSC and early progenitor cells might be harmful due to transgene toxicity and, potentially, induction of immune responses against the transgene product during the early engraftment phase in the context of a lymphocyte-sparing conditioning regimen. In order to target transgene expression to a desired lineage, we employ 2 strategies:

  • Transcriptional targeting: we continue to test and refine lineage-specific promoters
  • Posttranscriptional de-targeting: we harness microRNA activity to abrogate transgene expression in cells expressing by nature a specific microRNA. By incorporating multiple perfect target sequences for a microRNA into a vector, its expression becomes susceptible to negative regulation by that microRNA (see figure taken from Gentner and Naldini, Tissue Antigens, 2012;80:393-403). We identified two microRNA that show specific expression in HSC and early progenitor cells, miR-126 and miR-130a, and constructed “HSC-off” vectors that contain target sequences for miR-126 and/or miR-130a and thus lack expression in HSC as opposed to differentiated cells. These vectors allow delivering the transgene into HSC without altering their proteome, while benefitting from sustained multi-lineage expression in their progeny.

Transcriptional targeting positively regulates transgene expression (i.e., the promoter determines where the transgene is expressed), whereas miRNA de-targeting provides a negative control over transgene expression. Thus, transcriptional and post-transcriptional regulation complement each other and can be used in combination to yield highly regulated expression patterns. We are refining these combined targeting approaches for clinical development of gene therapy for several monogenic diseases including chronic granulomatous disease and globoid leukodystrophy.


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