Long term follow-up of pediatric patients affected by Hurler syndrome enrolled in a phase I/II ex-vivo gene therapy clinical trial and development of a novel ex-vivo gene therapy platform to treat lysosomal storage diseases

Lysosomal storage disorders (LSDs) are a group of genetic diseases characterized by errors of metabolism in lysosomal functions, resulting in accumulation of undegraded substrates, which causes multi-organ damage. Skeletal dysplasia is a severe and prominent feature of these diseases, characterized by a broad range of manifestations. Allogeneic hematopoietic stem cell transplantation (HSCT) often in conjunction with pre/peri enzyme replacement therapy (ERT) represents standard of care for some of them, e.g. Mucopolysaccharidosis I type Hurler (MPS-IH). Nevertheless, it is not able to fully address the skeletal manifestations. To overcome this limitation, hematopoietic stem/progenitor cell-gene therapy (HSPC-GT) represents a promising therapeutic strategy and previous data obtained by San Raffaele-Telethon Institute for Gene Therapy (SR-Tiget) demonstrated that HSPC-GT is capable of cross-correcting non-hematopoietic cells more effectively than ERT and HSCT. Over the course of my PhD program, I had the opportunity to focus on the long-term follow-up of 8 MPS-IH patients enrolled in a phase I-II clinical trial to prove safety and efficacy of HSPC-GT. In the project A of the present thesis, entitled “Long term follow-up of pediatric patients affected by Hurler syndrome enrolled in a phase I/II ex-vivo gene therapy clinical trial”, I investigated the preliminary neurological, skeletal and systemic outcomes (ocular, otorhinolaryngological, carpal tunnel syndrome and cardiological outcomes) to better characterize the progress of the MPS-IH typical manifestations after HSPC-GT. Furthermore, an initial retrospective comparison with the corresponding clinical outcomes of external cohorts of patients who received HSCT was performed with the aim to quantify the severity of the manifestations and the surgical burden after treatment. Overall, the interim results of the neurological analysis suggested biochemical detoxification in the CSF, indicating migration of gene-modified cells across the blood brain barrier and local engraftment in the CNS, and initial clinical response in terms of motor and cognitive abilities up to 4 years after treatment. The skeletal analysis showed no progression of skeletal dysplasia at multiple sites in all patients, both in terms of clinical, functional and radiological findings up to a median of 3.78 years after HSPC-GT. Moreover, our results in terms of systemic outcomes indicated stability or improvement of corneal clouding, hearing loss, cardiac manifestations and carpal tunnel syndrome in all patients after HSPC-GT up to 4 years after treatment. In summary, despite the short length of follow-up, the detailed set of outcomes data examined and the initial comparison to patients treated with HSCT with similar baseline characteristics suggested an early beneficial effect of HSPC-GT on the MPS-IH typical manifestations. The clinical outcomes, together with the analysis of safety and biological correction, supported the potential superiority of HSPC-GT over HSCT in correcting skeletal manifestations of LSDs, strengthening the rationale for the development of similar GT strategies in other rare LSDs with skeletal involvement and unmet medical need. Therefore, the project B of the thesis, entitled “Development of a novel ex-vivo gene therapy platform to treat lysosomal storage diseases”, is focused on the development of a new strategy consisting in a platform model where simultaneously three selected LSDs (Mucopolysaccharidosis type IVA, Mucopolysaccharidosis type IVB and Alpha Mannosidosis) can be treated using the same “platform”. This platform model will share the same mechanism of action (cross-correction of non hematopoietic cells by high levels of therapeutic enzyme delivered locally by hematopoietic cells), same platform manufacturing process of the key starting materials and of the final drug products, optimized platform of non-clinical GLP studies in line with 3Rs principles, an innovative platform clinical study protocol where the selected LSD products are simultaneously tested, and regulatory innovative aspects to move from the “1-to-1 sequential” drug development to the “simultaneous and parallel” development approach. For all the three diseases, our in vitro data, using patients’ derived cells, showed that the progeny of mobilized peripheral blood (mPB) CD34+ cells transduced with lentiviral vectors (LVs) produce supraphysiological levels of the respective enzymes, which can be internalized by patients’ cells of non-hematopoietic origin restoring the missing enzymes expression. The supraphysiological levels of enzymatic activities, obtained in our in vivo experiments, implied that the progeny of the engrafted gene-corrected HSPC could sustain local cross-correction of tissue resident non-hematopoietic cells. Furthermore, our study generated the preliminary proof-of-concept in vitro and in vivo data to support the preclinical development of HSPC-GT using the LV enhanced GLB1 (LV-enhGLB1) in a mouse model of β-GAL deficiency with the final goal of implementing the clinical trial of HSPC-GT for Mucopolysaccharidosis type IVB patients. In conclusion, the promising results of in vitro and in vivo analyses in all the three diseases, together with the significant therapeutic benefits observed in MPS-IH patients applying the same mechanism of action and therapeutic approach and using a lentiviral vector with the same structure and regulatory elements to drive supranormal expression of lysosomal enzymes, strongly support clinical testing of HSPC-GT in other LSDs patients.