Gene therapy is a broad term that refers to modification of a cells genetic material to induce a therapeutic effect. Strategies range from gene augmentation to genome editing.
Gene augmentation (aka gene addition) is the most commonly employed strategy for the treatment of Patients with inherited retinal degenerative blindness. This strategy typically relies on the use of a viral vector to deliver the gene of interest to the target cell type. The most commonly used virus for delivery of genes to retinal cells is the adeno associated virus (AAV).
AAVs are small nonpathogenic viruses that can carry genes up to ~5kb in size. Of the 105 different genes that we have found to cause disease in our patient population nearly 70% are small enough to fit into an AAV vector.
For genes that are too large to fit into AAV several other viruses, such as Adenovirus, which have the capacity to carry even the largest genes, are being developed.
AAV5 transduction induces robust GFP expression in human photoreceptor cells
Genome editing (AKA gene editing or genome engineering) is a term that is typically used to describe modification of a cells genetic material. This could include inserting, deleting and or replacing DNA.
Non homologous end joining (NHEJ) occurs when a double strand DNA break is made in the absence of a repair template. This DNA repair process is imprecise, typically resulting in creation of insertions and/or deletions that result in a shift in the reading frame and insertion of a premature stop codon.
As shown above, this strategy is ideal for preventing expression of a dominant disease causing allele such as Pro35His rhodopsin. Nucleotide and amino acid sequences of unaffected (P23) and affected (H23) rhodopsin alleles (A). Underlined sequences indicate guides targeting each allele. Red text indicates mutation (c.68 C > A transversion at codon 23) lying in the seed region of each guide. Representative gel image of T7E1 assays for CRISPR guide tested (B). Histogram showing percent NHEJ in cells transfected with each guide. Allele specific targeting can be increased by 19 and 22 fold respectively when the guide is designed such that the mutation falls within the seed region (C). Representative gel image of T7E1 assays in patient-derived iPSCs (+/P23H) and iPSCs from an unaffected individual (+/+) transfected with plasmids expressing the sgH23-2 guide and SaCas9, showing targeting of the mutant rhodopsin allele only (D).
Genome editing strategies
typically rely on the use of an
enzyme known as a nuclease
(eg., zinc finger nucleases,
meganucleases, Cas9 nucleases, etc.) to create a double stranded DNA break at specific locations within a cells genome. These breaks are subsequently repaired via either non-homologous end joining or homology directed repair.
Double Strand Break
Homology directed repair (HDR) occurs when a double strand DNA break is repaired using a donor template containing the wildtype (i.e., normal) sequence. Unlike NHEJ, HDR is precise and can be used to correct disease causing genetic mutations. HDR is the preferred strategy when correcting stem cells for autologous
cell replacement or evaluating disease pathology. We have successfully used this strategy to correct disease causing genetic mutations in dozens of different genes. For instance, here we are
demonstrating that HDR mediated correction of c.119-2A>C restores expression of normal NR2E3 transcript in patient-derived retinal organoids. A–F: Bright-field images of control (A, D), Patient 1 (B, E) and CRISPR-corrected Patient 1 (C, F) iPSCs (A-C) and retinal organoids (D-F). G: NR2E3 transcript analysis by semi-quantitative PCR in control and Patient 1 iPSC derived retinal cells before (-) and after (+) CRISPR correction demonstration restoration of wildtype transcript at 9 and 14 weeks following initiation of differentiation.