Despite the invaluable therapeutic benefits that gene therapy can offer, virus-mediated gene transfer can present significant risks. The potential for provirus integration to dysregulate nearby genes and create dangerous clonal outgrowths has been observed in certain clinical studies (Hacein-Bey-Abina et al. Science, 2003) and thus warrants additional measures to research and improve upon the safety of this treatment. In order to better understand the safety profile of various retrovirus vectors, the Adair lab has worked to develop novel methods of Retroviral Integration Site (RIS) analysis to carefully track gene-modified clones (Beard et al. Methods in Molecular Biology, 2014). This method combines the latest in next-generation sequencing technology with random genomic DNA shearing to reduce bias introduced by previous clone tracking techniques. While RIS is the most common clone tracking technology being applied to study blood cell biology in vivo, it is not quantitative and is biased by base pair content of the genomic sequence of interest and the degree of annotation of the host genome. To adapt clone tracking to more basic studies of hematopoiesis, the Adair lab applies retrovirus DNA barcoding to more quantitatively track gene modified cells in vivo.
In sum, RIS analysis identifies genomic loci where retroviruses integrate within the host genome, analyzes the effect of integration on nearby genes and permits continuous monitoring of specific clones throughout the remainder of follow-up. This reveals a wealth of information about the gene-modified clones including but not limited to their longevity, level of contribution, and selectivity under a variety of conditions.
In addition to methods generated in our laboratory, we collaborate closely with the laboratory of Dr. Grant Trobridge to develop bioinformatics pipelines that can process and streamline identification of meaningful clonal data from high throughput sequencing files. These platforms can be used to monitor clonal dynamics in individual subjects over time, across multiple subjects and species treated with the same vectors or across different vector types including gammaretrovirus, lentivirus, foamy virus and alpharetrovirus vectors to compare patterns of genomic integration. More recently we have applied this technology with new data outputs such as Circos to visualize how different ex vivo manipulations such as mobilization strategies and treatment with Rapamycin can affect integration patterns of the same lentiviruses (see our Publications page for more details).
Current research in the Adair lab applies RIS tracking of lentivirus gene modified cells to identify genes involved in early hematopoiesis. It is known that lentiviruses preferably integrate into actively transcribed genes. By tracking lentivirus integration sites in multiple blood cell lineages over long periods of follow-up in vivo, we can learn which genes were most likely active in long-term repopulating cells with multi-lineage potential. Here we apply RIS analysis in clinically relevant large animal models including the non-human primate (pigtail macaque; M. nemestrina) and patient data from our active clinical trials in gene therapy to identify these genes.
While RIS analysis is necessary for establishing the safety of vector-mediated gene therapy and can provide insight into biologically relevant genomic loci in different cell types, it is fraught with bias and as such, is not a robust method for quantitating hematopoiesis. This bias is driven by the different content and lengths of genomic fragments sequenced in each clonal pool, as well as by the ability to successfully align each sequence with a single genomic locus in the genome of interest. Proviral barcoding removes much of this bias. In this technique, the integrating portion of a retrovirus vector is engineered to contain a small (20-30bp) fragment of random nucleotides, flanked by known sequences in which to see primers. Thus, each amplified fragment is the same length and does not require genomic alignment to assign identity.
In collaboration with the laboratory of Dr. Matthew Porteus, we designed a barcoded self-inactivating lentivirus library which contained approximately 1.2 million different DNA barcodes. Currently we are applying this library to gene modified hematopoietic stem cells of nonhuman primates to compare clonal repertoires identified by RIS- and barcode-based methods. Current research in the Adair lab includes development of additional libraries which could be applied in competitive transplantation strategies to better quantitate differences in engraftment and reconstitution. These studies will help establish parameters to improve both cell and gene therapy approaches.