Ex vivo gene therapy with lentiviral vectors (LVs) is an established strategy to treat several genetic blood stem cell diseases. One such example is the gene therapy for metachromatic leukodystrophy (MLD), developed at SR-Tiget and approved in both Europe and the United States.

However, this strategy requires ex vivo manipulation of blood stem cells and patients’ conditioning with chemotherapy prior to their re-infusion, which represent significant hurdles that could be overcome with gene delivery in vivo.

In a paper just published in Nature, the group coordinated by Luigi Naldini, Director of the San Raffaele Telethon Institute for Gene Therapy (SR-Tiget) and Full Professor of Histology at the Vita-Salute San Raffaele University, and Alessio Cantore, group leader in the Liver Gene Therapy laboratory at SR-Tiget, identified an early postnatal window that enables LV-mediated gene delivery in circulating hematopoietic stem and progenitor cells in vivo.

The study opens new avenues for treating some genetic blood disorders without the need for stem cell transplantation or chemotherapy.

The study

The authors identified a population of circulating blood stem and progenitor cells which levels were higher in newborn (0-1-day old) mice as compared to older (1-, 2- and 8-week-old) mice. Presumably, these circulating blood stem cells trafficked from fetal-derived stem cell niches, still present in the postnatal liver and spleen, to the bone marrow.

“The identification of an early time window in which hematopoietic stem and progenitor cells are still circulating in the blood provides a unique opportunity for achieving the highest efficiency of LV-mediated transduction in vivo”, comment Doctor Michela Milani, first author of the study.

Systemic delivery of LVs via intravenous injections led to limited but reproducible transduction of hematopoietic stem cells in vivo. As evidenced by clonal tracking, transduced stem cells were able to generate multilineage blood cells (myeloid, B and T cells) – an ability that was retained when the transduced cells were transplanted in secondary host mice, in which they (and their progeny) also engrafted in the long term.

Levels of circulating hematopoietic blood stem and progenitor cells declined with age, making LV-mediated gene delivery less efficient with time.

However, forced mobilization of these stem cells with granulocyte-colony stimulating factor, combined with the drug Plerixafor (antagonist of the CXCR4 receptor), a strategy commonly used in the clinic to harvest stem cells, improved the efficiency of LV transduction not only in newborn but also in older mice, thus improving the efficiency and extending the time window for intervention in vivo. Moreover, efficiency of transduction was further improved with the use of lentiviral constructs that were shielded from phagocytosis.

Overall, the combination of these strategies allowed reaching a proportion of transduced stem cells in vivo that was never reported before and could provide therapeutic benefit if achieved in patients suffering from certain genetic conditions.

 

A promising tool for in vivo  correction of genetic diseases

The potential benefits of this in vivo gene therapy were thus tested in mouse models of three different genetic diseases: ADA-SCID, which is due to adenosine deaminase deficiency leading to severe combined immunodeficiency, autosomal recessive osteopetrosis, an inherited bone disease characterized by impaired bone resorption, and Fanconi Anemia, a rare genetic disorder caused by defective DNA damage repair.

In vivo LV-mediated gene delivery corrected ADA-SCID in newborn mice and led to prolonged survival and partially improved bone remodeling in osteopetrotic mice.

In Fanconi Anemia, where genetic correction endows hematopoietic stem cells with a selective advantage over mutated ones, in vivo gene transfer resulted in progressive expansion of the corrected cells up to full reconstitution of hematopoiesis with functional cells as well as prevention of bone marrow failure.

“Our results provide a proof of concept that in vivo gene therapy can be feasible if delivered in early postnatal phases, thanks to the permissiveness to lentiviral transduction of circulating hematopoietic blood stem cells that are homing to the bone marrow.”, comments Doctor Alessio Cantore.

“Importantly, we identified trafficking hematopoietic stem and progenitor cells also in the blood of newborn humans for the first few months of post-natal life, with levels that decline with age, similarly to what observed in mice. These results support future studies aiming to assess the translational potential of in vivo blood stem cell gene transfer in infantile patients”, concludes Professor Naldini.