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Human Stem Cell Gene Therapy – Safe and Effective



Two recent studies in Science reporting the outcome of lentivirally-mediated gene correction in hematopoietic stem cells (HSCs) to treat human patients have potentially ushered in a new era of safe and effective gene therapy.   Herein, we report on these exciting new clinical findings, both from the laboratory of Luigi Naldini at the San Raffaele Scientific Institute, Milan, Italy, on the treatment of metachromatic leukodystrophy (MLD), caused by mutations in the arylsulfatase A (ARSA) gene, and Wiskott-Aldrich syndrome (WAS), caused by mutations in the gene encoding WASP. 

MLD is an autosomal recessive inherited lysosomal storage disease (Aicardi 1998) which leads to progressive motor and cognitive impairment and death within a few years of the onset of symptoms.   Biffi and Montini et al started by isolating bone marrow derived CD34+ cells from three pre-symptomatic MLD patients (MLD01, 02 and 03).   These were transduced at high efficiency with LVs encoding the human ARSA cDNA and re-infused after myeloablation without immunosuppressive agents between 2 to 12 months before the reported age of onset of the disease in an affected sibling.   ARSA activity was reconstituted at greater than 10 fold the levels measured in healthy controls and no abnormal expansion or clonal outgrowth was detected in the peripheral blood (PB) and bone marrow.   One month after transplant, high-level stable engraftment of transduced cells in the bone marrow and peripheral blood of all patients was observed with between 45%-80% of colonies outgrowing from bone marrow samples harbouring the LV genome.   Accordingly, ARSA activity rose to above-normal values in therapeutically relevant myeloid populations and above normal levels of ARSA protein were isolated from hematopoietic cells after one month and cerebrospinal fluid (CSF) one to two years after transfusion, where before no ARSA was seen.   This is important as homing of HSPCs and their myeloid progeny to the CNS and PNS is therapeutically important.   Patient evaluation at time points beyond the expected age of disease onset demonstrated evidence of normal continuous motor and cognitive development compared to their untreated siblings.   The sibling of MLD01 was wheelchair-bound and unable to support their head and trunk at 39 months, but excitingly, after treatment patient MLD01 was able to stand, walk and run at 39 months of age and showed signs of continuous motor and cognitive development.   To the authors knowledge this is the first report of an MLD patient at 39 months displaying such positive clinical features.   Lastly, and perhaps most importantly, there was no evidence of cell clones harbouring integration sites at proto-oncogenes undergoing in vivo expansion or selection, while there was evidence of self-renewal and multi-lineage potential of the transduced engrafted HSCs, indicative of an abundant pool of engrafted transduced non-tumourigenic self-renewing progenitors. 

WAS is an inherited X-linked primary immunodeficiency syndrome which leads to infections, microthrombocytopenia, eczema, autoimmunity and lymphoid malignancies (Notarangelo et al and Catucci et al).   Following on from preclinical in vivo studies in WASP-deficient mice (Scaramuzza et aland Marangoni et al), Aiuti, Biasco and Scaramuzza et al report on the phase I/II clinical trial for treatment of human patients using ex vivo correction of autologous human HSPCs.   In this second study, CD34+ cells were collected from three patients with no compatible allogeneic donors and transduced twice with the corrective lentivirus (with 88-100% efficiency) and reinfused intravenously (~11 million cells ) three days after collection.   Analysis found evidence for robust multilineage engraftment of gene-corrected cells in bone marrow and peripheral blood up to the last assessment 30 months later, while WASP expression increased with time in most blood cells at a level comparable with normal donors, except for B cells and platelets, where expression was lower.   Although serious adverse infectious events occurred in two patients, overall clinical improvement resulted in a reduced disease severity score in all patients.   None of the three patients demonstrated signs of abnormal cellular expansion and platelet counts rose, although unfortunately not to normal levels.   Again, no evidence for adverse effects after lentiviral integration was observed, with insertional patterns between treated patients similar but different to sites found in the WAS retroviral gene therapy trial (Boztug et al), which showed a profile skewed towards transcription start sites.   Lentiviral insertion sites covered a range of gene ontologies, whereas retroviral insertions were more often associated with haematopoiesis-associated genes.   Common insertion sites (CIS) analysis identified well known hotspots in preclinical studies of lentiviral vectors (Biffi et al and Cattoglio et al) which are not identified with clonal expansion, dissimilar to retroviral use, and overall results indicated a lack of evidence for clonal expansions associated with CIS in proximity of known proto-oncogenes.

Simply put, the authors have presented a strategy for ex vivo gene correction in HSCs for inherited disorders which works and appears safe in comparison to previous strategies.   Long term analyses will undoubtedly need to be intensely scrutinised, but this research surely represents a huge step forward in the safe treatment of these and similar genetic disorders.



  • Aicardi, J. et al. Diseases of the Nervous System in Childhood (Mac Keith Press, London, ed. 2, 1998)
  • Biffi, A. et al. Lentiviral vector common integration sites in preclinical models and a clinical trial reflect a benign integration bias and not oncogenic selection. Blood 117, 5332–5339 (2011)
  • Boztug, K. et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N. Engl. J. Med. 363, 1918–1927 (2010)
  • Cattoglio, C. et al. Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood 110, 1770–1778 (2007)
  • Catucci, M. et al. Autoimmunity in Wiskott-Aldrich syndrome: An unsolved enigma. Front Immunol 3, 209 (2012)
  • Marangoni, F. et al. Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models. Mol. Ther. 17, 1073–1082 (2009)
  • Notarangelo, L. D. et al. Wiskott-Aldrich syndrome. Curr. Opin. Hematol. 15, 30–36 (2008)
  • Scaramuzza, S. et al. Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome. Mol. Ther. 21, 175–184 (2013)


From Science.

Stem Cell Correspondent Stuart P Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.