Our group is working on gene therapy for rare diseases and cancer. Retroviral/Lentiviral vectors are among the most efficient platform for the permanent integration of exogenous sequences into the genome of target cells for clinical gene therapy. Wild-type retro/lentiviruses and derived vectors integrate semi-randomly in the genome of the infected/transduced cell. The integration preferences of the proviruses are different according to the viral type and to the host conditions. Viral integrations are stably preserved by the host cells and are inherited by their progeny. However the insertion of exogenous material into the target genome can cause severe perturbations in the normal regulation of gene expression ultimately resulting in cellular transformation and cancer.
Safety monitoring of gene therapy clinical trials
We have a decade long expertise on the analysis of the interactions between viral vectors and the host genome to monitor insertional mutagenesis events and to assess the safety of gene therapy clinical trials. Our laboratory provides a facility (SAFEty of GENetic Engineering – SAFEGENE lab) for the safety monitoring of the clinical trials conducted in the Gene Therapy program at Dana-Farber/Boston Children’s Cancer and Blood Disorders Center and in other centers. Our work is to track the viral vector integrations on human specimens to monitor the stability of the engineered cells in patients after gene therapy. The SAFEGENE lab makes use of dedicated laboratory spaces and instrumentations for the processing of patient material in molecular contaminant free environment, access to high throughput sequencing facilities and customized bioinformatics platforms for integration sites annotation, analysis and storage.
Clonal tracking studies of engineered cells in animal models and humans
Upon genetic engineering with retroviral/lentiviral vectors each gene-modified cells becomes univocally marked by a viral integration site. Therefore viral insertions can be used as stable molecular barcodes to track the fate, survival and activity of virtually all types of genetically modifiable cells, from hematopoietic stem cells to neurons. The high diversity of marking reachable with insertional tagging makes this tool relevant to a variety of animal studies while the use of integrating viral vectors in the clinic makes this approach applicable to human studies differently from all the other available barcoding technologies. Our lab is currently conducting a clonal tracking study of T cells in patients with primary immunodeficiencies after gene therapy. These individuals are capable of producing new genetically engineered T cells several years after treatment even in the apparent absence of bone marrow progenitors. Through insertional tracking we are creating novel models of T cell clonal evolution in vivo in humans uncovering the existence of not yet described long living lymphoid-committed hematopoietic progenitors in the thymus. The results of this study could have a profound impact on the design of future strategies for the T cell engineering for immunodeficiencies and cancer immunotherapies. Another project is based on the tracking of hematopoietic stem/progenitor cells (HSPC) after transplant. We are applying mathematical models to track the activity of individual HSPC subtypes purified from the bone marrow of patients after gene therapy for hematological disorders. By this study we are unraveling, for the first time directly in humans, the changes in the composition of the repertoire of bone marrow derived CD34+ cells after transplant while addressing key biological questions regarding the shape of the human hematopoietic hierarchy. We are currently extending this study to transplantation procedures utilizing different sources of human HSPC including mobilized peripheral blood. The plasticity of HSPC make them a suitable target for genetic engineering also in the context of diseases that affect the brain. We are currently collaborating with the group of Dr. Alessandra Biffi to study how gene-modified HSPC give rise to brain microglia under physiological or stressed conditions and to assess their regenerative potential upon direct intracranial injection or through migration from the bone marrow.
Retroviral scanning for the discovery of cell-specific genetic signatures
Another area of research of our group involves the exploitation of the genome- and cell-specific nature of viral integration sites distribution as a novel tool called retroviral scanning for the identification of genomic features. Others and we have shown that a high degree of overlap is observable between viral integration sites and transcriptional/epigenetic cell-specific annotations. Compared to standard transcriptome profiling, retroviral scanning allows a more unbiased and less gene-oriented investigation of genomic rearrangements upon cell differentiation extending the detection range to other previously overlooked genomic areas (as the ones containing lncRNAs) governing cell differentiation processes. Notably, given the fact that viral integration site selection is dependent on the nuclear architecture of the target cell, this technique has the unique capability to detect at high-throughput level changes in topological nuclear localization of genomic areas along differentiation, an information not achievable by CAGE, RNA-seq or ChIP-seq. We are using different viral and non-viral integrating platforms to transduce individual human cell subtypes at different stages of maturation and identify specific insertional signatures marking chromatin and nuclear states of target cells. By high-throughput single-cell approaches we are correlating integration distributions with the transcriptome of engineered cells to unravel novel univocal marker of cell differentiation. With retroviral scanning we aim also at identifying genomic regions accessible to gene editing platforms like CRISPR/Cas9, pinpointing to loci potentially prone to host off-target activity as well as highlighting areas for safe and differentiation-specific targeted gene transfer.