Lentiviral gene therapy for hunter syndrome and pompe disease: Next generation therapies

Over the past several decades, Hematopoietic Stem and Progenitor
Cell-mediated Lentiviral Gene Therapy (HSPC-LVGT) has been
explored as a treatment for various inherited disorders, including
metabolic diseases. The enduring clinical benefits reported in numerous
trials have encouraged our laboratory to commence the Pompe and
Hunter gene therapy projects, where we aimed to examine the
therapeutic potential of HSPC-LVGT for the treatment of these two
diseases. This thesis encloses the resulting preclinical findings,
representing the foundation for future clinical applications of this
approach.
In the General Introduction, we provided a concise overview of
the history of gene therapy and the most significant steps toward the
development of effective treatments. We then elaborated on the concept
of virotherapy, discussing the viral vector systems most used in clinical
settings, with a focus on the molecular biology of lentiviral vectors. We
continued with an overview of the diseases commonly targeted by gene
therapy trials, the type of genes typically transferred, and the
phenomenon of cross-correction, which underlines the correction
mechanism during HSPC-LVGT for lysosomal storage disorders
(LSD). In Chapter 2, the study focused on the efficacy of tagging GAA
with IGF2 during HSPC-LVGT for treating murine Pompe disease. The
fusion of IGF2 to GAA significantly improved the therapy's efficacy
across various tissues, including skeletal muscles, heart, and brain, with
the brain exhibiting the most remarkable response. IGF2.GAA gene
therapy improved correction of glycogen accumulation, periodic acid-
Schiff (PAS)-related pathology and neuroinflammation. Furthermore,
correction of pathology was dose dependent, and occurred at a
clinically acceptable vector copy number (VCN). Chapter 3 elaborated
on the establishment of immune tolerance through HSPC-LVGT. The
study demonstrated that lentiviral gene therapy can prevent the
formation of neutralizing antibodies against the transgene, and that
ineffective HSPC-LVGT can be safely supplemented with Enzyme
Replacement Therapy (ERT). Moreover, the presence of HSPC-derived
cells expressing GAA in the thymus indicated the establishment of
central immune tolerance, preventing the formation of neutralizing
antibody also against the intravenously infused protein. In Chapter 4,
we investigated the effects of tagging IDS with IGF2, ApoE2 and
RAP12x2 on the efficacy of HSPC-LVGT in treating a murine model
of Hunter disease. We found that IGF2, ApoE2, but not RAP12x2,
caused a dose-dependent improvement of correction of brain
pathology. The tag-mediated improved efficacy was independent of the
number of brain-engrafted cells, a general mechanism of correction
during HSPC-LVGT, but correlated with plasma levels of tagged IDS
protein. This potentially implied a tag-mediated protein delivery to the
brain as an additional mechanism to cell engraftment of donor-derived
cells. Chapter 5 assessed the effects of IDS tagging in correcting
peripheral pathology of a murine Hunter disease model. Gene therapy
with tagged IDS caused correction of pathology at levels that were
comparable to untagged IDS, resulting in normalization of
abnormalities in multiple peripheral tissues, including liver, spleen,
kidney, heart valves, and bone microarchitecture. However, the
treatment was ineffective in reliving pathology in cartilage tissue,
irrespective of the vector employed. Importantly, IDS.IGF2co gene
therapy was exceptionally effective in treating pathology of the great
hart vessels. We also collected evidences suggesting that correction of
pathology in peripheral tissues involved secretion of IDS protein into
the bloodstream and uptake into target tissues, in addition to the
engraftment of donor-derived cells. In Chapter 6, we introduced IGF2
tag variants with the goal to enhance the reach and the safety of IGF2-
based therapeutics. The new IGF2 variants were characterised by a
substitution of domain C and parts of domain A and B of the mature
human IGF2 with epitopes capable of targeting receptors different from
those typically engaged by IGF2. The resulting IGF2indel versions with
ApoE2, RAP12x2 and ApoB inserts could not bind the IR-A,
previously proposed to be involved in hypoglycaemia events after
intravenous injection of a IGF2-tagged protein, and bound LRP-1 with
high affinity. These data point at the IGF2indel design as promising
tool for enhancing safety and the reach of IGF2-based therapeutics. In
the General Discussion we reflected on the key achievements of the
thesis, and we discussed about some aspects of the vector design that
could benefit some refinements. Next, we rationalized about the
mechanisms of correction of brain pathology after gene therapy, as
well as the limitations of the treatment in correcting the cartilage
pathology. We also reflected on which factors affect the genotoxic
potential of lentiviral vectors.
These findings signify a critical step towards effective
treatments for Pompe disease and Hunter disease, and demonstrate that
protein-tagging can improve the efficacy of this treatment. While
continuous efforts to refine these vectors to further enhance safety and
efficacy are recommended, this research demonstrates their potential to
significantly improve the quality of life of patients.