The Problem: Mitochondria generate energy within each and every human cell. Mutations to the mitochondrial DNA, whether inherited or acquired over a lifetime lead to metabolic, neurodegenerative and cardiovascular diseases of aging.

The SENS Research Foundation has formulated seven practical ‘repair’ strategies to the common drivers of aging. Whereas some of these strategies are now widely researched by the scientific establishment the MitoSENS strategy for dealing with mitochondrial damage is among the most novel. Our theory is, through “allotopic expression”, that is by placing functional copies of critical mitochondrial DNA (mtDNA) genes in the nucleus of the cell one could alleviate defects arising due to mutations in mtDNA.

When it was proposed, this unique and ambitious strategy was perhaps too ‘daring’ for mainstream labs and funding agencies to contemplate. Consequently, the MitoSENS approach has been an ‘in house’ project for SENS that would not have been possible without community support. So far, this community-funded approach has an excellent track record leading to groundbreaking discoveries:

In 2013 SENS organized its first crowdfunding campaign specific to MitoSENS in partnership with LongeCity. The small initiative seeded significant research momentum and paved the way for a larger fundraiser in 2015 at Lifespan.io. Breakthrough discoveries followed and a proof-of-principle for the MitoSENS approach was established for the first time in human cells. Here, the MitoSENS team in collaboration with leading scientists Prof. Martin Brand and Dr. Birgit Schilling from the Buck Institute showed that allotopic expression of two mtDNA genes could bring back several functions in a patient cell line with a severe mutation in one of the mtDNA genes namely ATP8.

To move this strategic advancement toward the clinic, SRF then created the “maximally modifiable mouse model”. This mouse has a unique modification in their nuclear genome to allow a targeted insertion of new genes at a specific location. Using this mouse, we are ready to take the next step and pursue mitochondrial gene therapy in an animal model.

Project

Mice of the C57/BL6MT-FVB strain (let’s call them “SickMice”) have a mitochondrial gene defect (a mutation in the mitochondrial ATP8 gene) and exhibit several age-related symptoms including lower fertility, arthritis, type II diabetes and neurological impairments. Since mitochondria are only inherited from the mother, cross-breeding female SickMice with male mice from other models will result in the same mitochondrial dysfunction.

We will use the maximally modifiable model to create a new transgenic mouse (the “allotopic ATP8 transgenic mouse – Mitomouse”). This mouse will have the ATP8 gene that is important for mitochondrial function ‘hidden’ in the cell nucleus and thus capable of being passed on to offspring irrespective of gender.

Our hypothesis is that both male and female offspring from SickMice x MitoMice will result in rescued mitochondrial function. This would prove the viability of the MitoSENS strategy by showing that functional backup copies of mitochondrial DNA genes in the nucleus can replace their mutated counterparts in live animals.

Finally,. Success here will

A large part of the cost will be in the generation of the new MitoMouse model. This will be subcontracted (Applied Stem Cells, Milpitas, California) as it is a very specialized technique that we do not have in house and also do not need in the future. Significant further expenses will be linked to maintaining sufficiently large colonies (both MitoMouse and SickMouse) to assess behavioral and physiological functions. We will collaborate with the world-leading Buck Institute (Prof. Martin Brand and Dr. Birgit Schilling), which has specific expertise in humane animal husbandry with regard to aging-related mouse studies. Additional costs will contribute to special reagents required for measuring oxygen consumption using Seahorse and Mass Spectroscopy for proteomics.

$50,000 — Initial Goal

Milestone 1: Creating the “MitoMouse”

We will generate a plasmid construct for the optimized mouse ATP8 gene, to express from the nucleus instead of the mitochondria. This construct will then be injected into embryos from “Maximally Modifiable” mice during the blastocyst stage. These embryos will be transplanted into surrogate mothers to generate “founder” mice with the transgene. They will be genotyped to validate this gene engineering and confirm nuclear integration of the allotopic gene.

Estimated study duration: ~6 months. Milestone 2: Examining offspring of SickMice & MitoMice

A cohort of “SickMice” (females) will be crossbred with “MitoMice” (males); aiming for at least 10 offspring. These offspring will then be validated at the genome level (homozygous/ heterozygous for the allotopic gene and homoplasmy/ heteroplasmy for the mtDNA mutation load), protein level (Western blotting), biochemistry (oxygen consumption analysis using Seahorse XF Analyzer & functional testing for mitochondria).

Estimated study duration: ~12 months. $65,000 — Stretch Goal 1

Milestone 3: Physiologically Testing Offspring Mice

The mitochondrial dysfunction in SickMice leads to observable frailty and behavioral changes. If this stretch goal can be reached, it would enable us to investigate whether these traits are improved/changed in live offspring: Age-related behavioral scoring (open field & gait analysis)

Endurance running – muscle function

Rescue of type II diabetes phenotype Estimated study duration: ~2 months additional. $75,000 — Stretch Goal 2

Milestone 4: Fertility Analysis

This stretch goal would enable us to keep a colony of MitoMice and offspring for further testing and sharing with other research groups. One of the most noted characteristics in SickMice is their altered fertility and the potential of rescued fertility can be conclusively investigated in this context.

Estimated study duration: ~4 months additional.

Amutha Boominathan, Ph.D.

Project Lead and MitoSENS Group Lead Dr. Amutha Boominathan is the Group Lead for the MitoSENS project at SENS Research Foundation, Mountain View, California. She has >18 years of postdoctoral experience in mitochondrial biogenesis and leads a highly motivated team of researchers at SENS Research Foundation in developing and advancing technologies to functionally relocate the mtDNA genes to the nucleus. She is passionate about biomedical research and would like to apply her expertise and experience in finding cures for age related diseases. Dr. Boominathan received her PhD in Biochemistry from the National Chemical Laboratory, India, in 1998. Caitlin Lewis

Research Associate Caitlin received her B.S. in Molecular Biology from San Jose State University in 2016, with minors in chemistry and business. During her undergraduate career she conducted research investigating the regulatory relationships between transcriptional co-activators and transcription factors common to developmental and oncogenic pathways. Caitlin is specifically interested in personalized medicine and targeted molecular therapeutics, and has joined SRF scientists with the goal of engineering improvements in human health. As a member of the MitoSENS team, her work includes the allotopic expression of mitochondrial genes for restoration of normal respiratory chain function. Bhavna Dixit

Research Associate Bhavna Dixit earned her Masters in Biotechnology from Amity University, India in 2012. As a Research Scholar at the National Research Center in Plant Biotechnology, India, she worked on gene silencing and transcriptional analysis of pathogenicity genes in wheat leaf rust fungus. She moved to California in 2015 and completed a Professional Certificate Program in Biotechnology in March 2016 from UCSC Extension, Silicon Valley. After working as a volunteer with SRF for 6 months, she became a MitoSENS researcher and is working on the allotopic expression of mitochondrial genes.