Vectorology - GEG Tech top picks

It is a pity that non integrating lentiviral vectors are unknown or underused. This is not THE solution but in several cases, I think their use would be more relevant than AAVs use in term of immune response. The advancements of gene therapy will go through the design and development of complementary vector platforms.

In a recent study, a team of researchers used a CRISPR/Cas9 system to evaluate the use of tRNA by deleting two tRNA genes from the genomes of hyperhepatocellular carcinoma and human chronic myeloid quasi-haploid leukaemia cells. The authors discovered numerous unexpected genomic changes at the target region using an improved droplet-based target enrichment approach followed by Oxford Nanopore Technology long-read sequencing. The method used in this study demonstrates that CRISPR/Cas9 can lead to the integration of endogenous and exogenous DNA fragments and also produce local inversions, duplications and insertions of functional target-derived fragments. This research presents evidence that a combination of duplication and inversion, as well as integration of exogenous DNA fragments and clustered interchromosomal rearrangements, can occur simultaneously. Furthermore, it was shown for the first time that the target-derived fragments were nevertheless functional despite these modifications, which may complicate mechanistic explanations. These results reveal a new example of unintended CRISPR/Cas9 editing events that can go unnoticed and have a significant impact on the conclusions drawn from experimental reads. 

Great publication in an internationally well-respected journal, about the integration of AAV genomes. This is a critical point because this type of event can induce genotoxicity effects which are harmful in gene therapy contexts. For far too long, this point has remained unclear, preventing its study and biasing the evaluation of the AAV biosafety level. This lack of clarity has also prevented comparisons with other viral vector options to design gene therapy strategies which would have been more relevant. For example, several publications showed very promising results with non-integrating lentiviral vectors in the field of eye gene therapy.

The issues of AAV integrations are even more important after the recent marketing authorizations of gene therapy products Luxturna and Zolgensma which are based on this type of viral vector.

In the mid-1990s, several years after a variety of viral vectors started being used for gene transfer into cells, tissues, and in some cases humans, it became clear that there were considerable limitations. For applications requiring a stable genetic modification that could lead to sustained gene expression in cells and their progeny, a delivery vehicle was needed that could transduce foreign cargo into dividing and nondividing cells, without causing immuno- or genotoxicity. A decade earlier, human immunodeficiency virus (HIV) had been identified as the cause of AIDS, and rapid studies of its biology led to the idea that this genus of retrovirus—lentiviruses—could be optimized for gene therapy.

The purpose of this white paper is to review the evidence of rAAV-related host genome integration in animal models and possible risks of insertional mutagenesis in patients. In addition, technical considerations, regulatory guidance and bioethics are discussed.

Although CRISPR/Cas9 mainly generates short insertions or deletions at the target site, it can also make large deletions of DNA around the specific target site. These large deletions pose safety concerns and can reduce the efficiency of functional editing. The WFIRM team is looking at ways to reduce the risk of this happening. The research described in their recent paper, published recently in Nucleic Acids Research, aimed to combat the generation of unpredictable long DNA deletions on target and find a way to prevent this, a key step towards the development of gene editing therapies to treat genetic diseases. The team evaluated a variety of human cells and genes of interest and found that fusing DNA polymerase I or the Klenow fragment to the Cas9 enzyme minimised large, unanticipated deletions of genomic DNA without sacrificing the efficiency of genome editing. On the contrary, it even increased editing efficiency in primary human cells.  

Bluebird bio has stopped two clinical studies of its gene therapy for sickle cell disease after one participant developed leukemia and researchers reported another has a cancer-like disease of the bone marrow. 

Bluebird is trying to determine where the vector integrated into the cell's genome, aiming to assess proximity to the genes that triggered the patient's cancer. The company will also examine whether the vector influenced gene expression, said chief scientific officer Phillip Gregory. 

Part of answer?

https://pubmed.ncbi.nlm.nih.gov/22523069/

https://pubmed.ncbi.nlm.nih.gov/19707188/

In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software.
The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions.
This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.