Moving Forward with the Maximally Modifiable Mouse

One of the past projects undertaken by the SENS Research Foundation was the groundwork for a better methodology of carrying out investigative gene therapies in mice. This is called the Maximally Modifiable Mouse, and it might be thought of as a sort of mirror image of CRISPR gene editing technology: instead of bacterial genetic mechanisms normally used to defend against viruses being adapted to insert DNA into cells, is is the case for CRISPR, in the Maximally Modifiable Mouse viral genetic mechanisms normally used to attack bacteria are adapted and placed into the mouse genome to act as a docking station for the later insertion of arbitrary genetic material.

The point of the exercise is that the Maximally Modifiable Mouse technology makes it possible, or at the very least easier and less costly, to make precise genetic alterations in mice at any point in life, young or old. Most research into cellular mechanisms involves genetic engineering at some point, even if the end result for human medicine is usually some other form of intervention. It is the most effective way, and sometimes the only way, to make progress in understanding the inner workings of specific cellular processes. This engineering is still largely accomplished through the creation of altered lineages of mice rather than the application of gene therapies to normal adult mice, however. Building those lineages takes time and money, and it might be possible to cut this cost from the picture via the Maximally Modifiable Mouse. Cheaper research is faster research, and that is one of the goals of this tool.

The other important goal here is to build a system that can be used to cost-effectively test therapeutic genetic alterations aimed at rejuvenation. The obvious candidate here is allotopic expression of mitochondrial genes, which requires genetic material to be delivered to the cell nucleus in order to bypass the consequences of damage to mitochondrial DNA. This is one of the root causes of aging, and allotopic expression has the potential to eliminate it. There will likely be other gene therapies to help with other forms of damage as this age of genetics moves on; perhaps the insertion of artificial enzymes capable of safely breaking down forms of metabolic waste that presently accumulate, for example. Almost any therapy that involves adding novel proteins or changing levels of existing proteins might in the future be accomplished with gene therapies at least as efficiently as via small molecule drugs – or at least once the research and development community has moved beyond its current reluctance regarding elective genetic alteration.

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.

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.

I’m pleased to see that the SENS Research Foundation, with funding from the Forever Healthy Foundation and other donors in our community, has started a collaboration with the Buck Institute for Research on Aging to move ahead with field testing of the Maximally Modifiable Mouse. Infrastructure projects with the potential to greatly reduce cost and time in research are one of the most important activities in any field of research. Few people pay enough attention to such work, and it rarely results in the headlines it deserves, but this sort of thing is what drives the pace of progress over the longer term.

SRF and Buck Institute to Collaborate on Gene Therapy

SENS Research Foundation (SRF) has launched a new research program focused on somatic gene therapy in collaboration with the Buck Institute for Research on Aging. Brian Kennedy, PhD, a leading expert on the biology of aging, will be running the project in his lab at the Buck. Many potential treatments of age related diseases require the addition of new genes to the genome of cells in the body, a technology known as somatic gene therapy. The technology has been hampered, up until now, by the inability to control where the gene is inserted. That lack of control resulted in a significant risk of insertion in a location that encourages the cell to become malignant.

SRF has devised a new method for inserting genes into a pre-defined location. In this program, this will be done as a two-step process, in which first CRISPR is used to create a “landing pad” for the gene, and then the gene is inserted using an enzyme that only recognizes the landing pad. SRF has created “maximally modifiable mice” that already have the landing pad, and this project will evaluate how well the insertion step works in different tissues. “Somatic gene therapy has been a goal of medicine for decades. Being able to add new healthy genes will enable us to address treatments of such age-related diseases as atherosclerosis and macular degeneration. Our collaboration with SRF will substantially move us toward finding effective treatments to genetically based age related diseases.”

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