An empowered, clinically viable hematopoietic stem cell gene therapy for the treatment of multisystemic mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (MPS II), or Hunter syndrome, is a rare X-linked recessive lysosomal storage disorder due to a mutation in the lysosomal enzyme iduronate-2-sulfatase (IDS) gene. IDS deficiency leads to a progressive, multisystem accumulation of glycosaminoglycans (GAGs) and results in central nervous system (CNS) manifestations in the severe form. We developed up to clinical readiness a new hematopoietic stem cell (HSC) gene therapy approach for MPS II that benefits from a novel highly effective transduction protocol. We first provided proof of concept of efficacy of our approach aimed at enhanced IDS enzyme delivery to the CNS in a murine study of immediate translational value, employing a lentiviral vector (LV) encoding a codon-optimized human IDS cDNA. Then the therapeutic LV was tested for its ability to efficiently and safely transduce bona fide human HSCs in clinically relevant conditions according to a standard vs. a novel protocol that demonstrated superior ability to transduce bona fide long-term repopulating HSCs. Overall, these results provide strong proof of concept for the clinical translation of this approach for the treatment of Hunter syndrome.

An innovative hematopoietic stem cell gene therapy approach benefits CLN1 disease in the mouse model.

Hematopoietic stem and progenitor cells (HSPCs) can establish a long-lasting microglia-like progeny in the central nervous system of properly myeloablated hosts. We exploited this approach to treat the severe CLN1 neurodegenerative disorder, which is the most aggressive form of neuronal ceroid lipofuscinoses due to palmitoyl-protein thioesterase-1 (PPT1) deficiency. We here provide the first evidence that (i) transplantation of wild-type HSPCs exerts partial but long-lasting mitigation of CLN1 symptoms; (ii) transplantation of HSPCs over-expressing hPPT1 by lentiviral gene transfer enhances the therapeutic benefit of HSPCs transplant, with first demonstration of such a dose-effect benefit for a purely neurodegenerative condition like CLN1 disease; (iii) transplantation of hPPT1 over-expressing HSPCs by a novel intracerebroventricular (ICV) approach is sufficient to transiently ameliorate CLN1-symptoms in the absence of hematopoietic tissue engraftment of the transduced cells; and (iv) combinatorial transplantation of transduced HSPCs intravenously and ICV results in a robust therapeutic benefit, particularly on symptomatic animals. Overall, these findings provide first evidence of efficacy and feasibility of this novel approach to treat CLN1 disease and possibly other neurodegenerative conditions, paving the way for its future clinical application.

Biodegradable polymeric nanoparticles administered in the cerebrospinal fluid: Brain biodistribution, preferential internalization in microglia and implications for cell-selective drug release.

Cell-selective drug release in the central nervous system (CNS) holds great promise for the treatment of many CNS disorders but it is still challenging. We previously demonstrated that polymeric nanoparticles (NPs) injected intra-parenchyma in the CNS can be internalized specifically in microglia/macrophages surrounding the injection site. Here, we explored NPs administration in the cerebrospinal fluid (CSF) to achieve a wider spreading and increased cell targeting throughout the CNS; we generated new NPs variants and studied the effect of modifying size and surface charge on NPs biodistribution and cellular uptake. Intra-cerebroventricular administration resulted in prevalent localization of the NPs in proximity to stem-cell niches, such as around the lateral ventricles, the subventricular zone and the rostral migratory stream. NPs internalization occurred preferentially in brain myeloid cells/microglia. We demonstrated that brain biodistribution and extent of internalization in microglia are influenced by NPs dimensions and can be improved by applying a transient disruption of the blood-brain barrier with mannitol, leading to NPs internalization in up to 25% of brain myeloid/microglia cells. A fraction of the targeted cells was positive for markers of proliferation or stained positive for stemness/progenitor-cell markers such as Nestin, c-kit, or NG2. Interestingly, through these newly formulated NPs we obtained controlled and selective release of drugs otherwise difficult to formulate (such as busulfan and etoposide) to the target cells, preventing unwanted side effects and the toxicity obtained by direct brain delivery of the not encapsulated drugs. Overall, these data provide proof of concept of the applicability of these novel NP-based drug formulations for achieving internalization not only in mature microglia but also possibly in more immature myeloid cells in the brain and pave the way for brain-restricted microglia-targeted drug delivery regimens.

Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells.

Recent evidence indicates that hematopoietic stem and progenitor cells (HSPCs) can serve as vehicles for therapeutic molecular delivery to the brain by contributing to the turnover of resident myeloid cell populations. However, such engraftment needs to be fast and efficient to exert its therapeutic potential for diseases affecting the central nervous system. Moreover, the nature of the cells reconstituted after transplantation and whether they could comprise bona fide microglia remain to be assessed. We demonstrate that transplantation of HSPCs in the cerebral lateral ventricles provides rapid engraftment of morphologically, antigenically, and transcriptionally dependable microglia-like cells. We show that the cells comprised within the hematopoietic stem cell compartment and enriched early progenitor fractions generate this microglia-like population when injected in the brain ventricles in the absence of engraftment in the bone marrow. This delivery route has therapeutic relevance because it increases the delivery of therapeutic molecules to the brain, as shown in a humanized animal model of a prototypical lysosomal storage disease affecting the central nervous system.

Brain conditioning is instrumental for successful microglia reconstitution following hematopoietic stem cell transplantation.

The recent hypothesis that postnatal microglia are maintained independently of circulating monocytes by local precursors that colonize the brain before birth has relevant implications for the treatment of various neurological diseases, including lysosomal storage disorders (LSDs), for which hematopoietic cell transplantation (HCT) is applied to repopulate the recipient myeloid compartment, including microglia, with cells expressing the defective functional hydrolase. By studying wild-type and LSD mice at diverse time-points after HCT, we showed the occurrence of a short-term wave of brain infiltration by a fraction of the transplanted hematopoietic progenitors, independently from the administration of a preparatory regimen and from the presence of a disease state in the brain. However, only the use of a conditioning regimen capable of ablating functionally defined brain-resident myeloid precursors allowed turnover of microglia with the donor, mediated by local proliferation of early immigrants rather than entrance of mature cells from the circulation.