Applied Stem Cell

 

Creation of a “Maximally Modifiable Mouse” 
We hope this project will demonstrate the feasibility of bona fide rejuvenation biotechnologies – therapies that remove, replace, repair or render harmless the pre-existing burden of cellular and molecular damage of aging in persons who have already suffered substantially from the degenerative aging process. It requires that new therapies be tested in animal models that have already undergone significant biological aging. Many of these therapies will be best demonstrated using gene therapy in animal models, and may ultimately require gene therapy for maximal efficacy in humans.
 
Conventional transgenic animals bear their novel genes in the germ line, and although convenient methods for inducing the expression of therapeutic transgenes late in life exist, doing so still requires the custom generation of a line of transgenic animal for each new tested gene, and then allowing it to age, typically for two or more years, before the induced transgene’s effects can be tested. This greatly slows down the development cycle of testing, refining, and iteratively re-testing therapeutic genes. Somatic gene therapy allows for much more rapid testing of therapeutic genes, but there are substantial limitations facing the existing methods of somatic gene therapy, even in mice. Plasmid methods lead to only transient gene expression, and repeated dosing cannot cumulatively increase gene dose per cell, or often increase the ratio of cells in a tissue expressing the gene. Existing viral somatic gene therapy vectors are often limited to small DNA payloads, and are generally prone to disrupting adjacent genes at the site of viral integration, leading to a significant risk of mutation.
 
A promising alternative is the use of integrases from bacteriophages (or “phages,”), a class of virus whose hosts in nature are bacteria. Phage integrases are enzymes that catalyze precisely-targeted, unidirectional recombination between paired DNA recognition sequences: one (attB) a specific site in the bacterial host where the viral DNA is inserted, and another (attP) in the phage genome, from which the viral DNA is copied. Moreover, phage integrases can be used to insert arbitrary amounts of DNA into the host genome. To exploit phage integrases for gene therapy in mammals, one plasmid is generated containing the gene(s) to be inserted linked to an attB site, and another is generated containing the phage integrase; the plasmid DNA is translated in the host cell, generating the integrase, which then inserts the attB-bearing gene of interest into the host genome, with essentially no risk of gene disruption; the attP and attB sites are both destroyed in the process.  The serine integrase from the mycobacteriophage Bxb1, in particular, is extremely precise: it will only mediate integration at specific attB sites. The Bxb1 integrase has already been demonstrated as a highly effective tool for somatic gene therapy in Drosophila, and has been shown to allow repeated, high-titer delivery of novel genes.
 
Unfortunately, mammals lack attP sites in their genomes, and thus the Bxb1 integrase cannot be used to insert new genes into mammalian model organisms such as the mouse. This limitation could be overcome with a one-time germline insertion of the Bxb1 insertion sequence into a transcriptionally-active but safe genomic location in the mouse genome: in such mice, the Bxb1 integrase system could be used at any time during the lifespan to insert therapeutic genes of any size, and with repeated rounds of gene dosing with multiple delivery methods to hit all the relevant tissues in the animals’ body, with only a very low risk of mutagenesis. The effects of such genes on age-related disease could then be rapidly evaluated, and if improvements need to be made, a new transgene constructed and tested immediately in mice who are already the same age, without having to wait for a new generation of transgenic animal to be generated, born, mature, and age with every round of testing.