Longer description of your proposed project

Summary: Tardigrades are microscopic extremophile animals, and are known for their tolerance of extreme stress (radiation, desiccation, solvents, etc.). Several genes have been identified as contributors to this stress tolerance. Published data on the expression of these genes in mammalian cells has led to promising (although mixed) results. Our own unpublished studies show that expression of one gene, MAHS, in human stem cells significantly increases cell survival after solvent, mechanical, and metabolic stress. In this proposal, we would like to synthesize “humanized” variants of MAHS targeting different parts of human cells, and test if survival can be improved through expression of single or multiple of the variants.

Funding summary: Request is only for the gene synthesis ($2500-$8000), transduction of the genes into human cells and testing will be performed by graduate students that have stipends covered by fellowship. This proposal will provide proof-of-concept data to leverage other funding (e.g. CIRM, NIH, NSF) for full characterization, testing, and translation to practice.

Motivation & Objective: Mammalian cells are increasingly used as tools in research, clinical practice, and bioproduction. Notable examples are the clinical use of stem cell grafts, CAR-T therapy, and the use of mammalian cells for the production of growth factors and antibodies. Despite the breadth of potential use, numerous challenges remain, including the inherent fragility of mammalian cells. Regardless of the ultimate use, stress is an unavoidable factor, with mechanical, toxic, oxidative, and metabolic insults decreasing yield and reducing effect. As a clinical example, one study involving the injection of cells into damaged cardiac tissue observed ~30% cell death 24 hrs post injection. In this proposal, we intend to synthesize “humanized” versions of the tardigrade gene MAHS for improved performance in human cells.

Long-term Goals & Benefits: This proposal is for basic engineering research and will not have a immediate societal impact. However, development of improved stress tolerance has significant potential near-term benefit (e.g. cheaper production of growth factors, injection of therapeutic cells) and mid-term benefit (e.g. increasing portability and access to useful human cell types in resource poor areas).

Background & Preliminary Data: The tardigrade gene MAHS has been shown to increase human cell survival after hyperosmotic stress. In our own unpublished studies, we observe significant increases in human stem cell survival after a range of representative stresses (Date figures available upon request). With MAHS expression, human ASCs are more resistant to mechanical stresses of injection, even in 34-gauge needles. Similarly, they show increased survival in depleted media, indicating resistance to metabolic stress. Finally, they also show improved tolerance to the solvent and common cryoprotectant DMSO. Overall, there is strong evidence that tardigrade gene expression is a viable strategy for increasing stem cell stress tolerance.

Approach: MAHS is targeted to the mitochondrial membranes in both tardigrades and humans. Targeting to other subcellular organelles may provide increased human cell stress tolerance that synergizes with the original MAHS. Specifically, we will design MAHS variants lacking the mitochondrial localization sequence, and include localization tags for 1) the nuclear membrane, 2) the endoplasmic reticulum, 3) the sarcoplasmic reticulum, and 4) the plasma membrane. Stress testing will be conducted, and promising variants will be selected for co-expression studies by synthesizing dual expression vectors (estimated 6 combinations). Genes will be synthesized by outsourcing to VectorBuilder.

Select References on MAHS:

Hesgrove C, Boothby TC. The biology of tardigrade disordered proteins in extreme stress tolerance. Cell Commun Signal. 2020 Nov 4;18(1):178.

Tanaka S, Tanaka J, Miwa Y, Horikawa DD, Katayama T, Arakawa K, Toyoda A, Kubo T, Kunieda T. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells. PLoS One. 2015 Feb 12;10(2).

Select Reference on Cell Stress:

Amer MH, Rose FRAJ, Shakesheff KM, Modo M, White LJ. Translational considerations in injectable cell-based therapeutics for neurological applications: concepts, progress and challenges. NPJ Regen Med. 2017;2:23.

Describe why you think you're qualified to work on this

The TIME Lab at UC Riverside is headed by Dr. Joshua Morgan, Assistant Professor in Bioengineering. Lab space includes all appropriate cell culture, microscopy, general lab equipment required to leverage the proposed gene synthesis project. All work will be conducted in BSL-2 facilities with appropriate institutional oversight. The TIME lab is successful at attracting funding, two nationally competitive awards we would like to highlight are:

National Science Foundation CAREER 2046093

Morgan (PI)

04/01/21-03/31/26

Mechanoaging: Tension mediated propagation of cellular senescence

American Heart Association Innovative Project Award 19IPLOI34760636

Morgan (PI)

07/01/19 - 06/30/21

Integration of tardigrade survival proteins to increase resilience of vascularized graft tissue in ischemic environments

Why the Morgan Lab at UC Riverside? We have generated a body of data demonstrating the robust protective effect of MAHS in human stem cells. Two of the graduate students in the lab have fully funded fellowships to work on these projects (one is funded by CIRM for incorporation of tardigrade genes into stem cells, the other is funded by NSF GRFP for studying tardigrade gene mechanism). We have generated dozens of cell lines with tardigrade gene expression. These are studies we are fully capable of completing, once we have the genes synthesized.

Why Josh Morgan? My doctoral, postdoctoral, and recent research has focused on elucidating the cellular and subcellular mechanotransduction pathways in the eye, lung and vasculature resulting in 37 peer-reviewed papers and one book chapter. The diversity of the published work reflects my integrative approach and reflects the breadth of experience essential for pursuing interdisciplinary work. This is demonstrated by the breadth of subject matter in published work, including in flagship publications of the national and international organizations ASCB, ISER, and ARVO as well as journals representing other fields such as physical chemistry and bioengineering, and has exposed the PI to expert perspectives in numerous subjects. Through these studies, I have gained extensive experience planning, performing, troubleshooting, and analyzing in vitro cell culture experiments with heterogeneous cell populations and appropriate mechanical (e.g. fluid forces, stretch, tension, etc.) and biological (e.g. extracellular matrix, growth factors, etc.) cues. My independent career at UCR has focused on developing physiologically relevant in vitro models for tissue, and investigating the roles of stress and mechanics in tissue-scale biology. Our interest in stress, and cellular mechanisms for tolerating stress, has led to the incorporation of tardigrade transgenes in human cells, and the collection of compelling preliminary data supporting this application. Overall, my record demonstrates the history of productivity and commitment to mentorship required to lead a successful independent research program. My experience and skills, reflected in my published work and preliminary data, are uniquely suited to pursuing the proposed research.

Full list of publications:

https://scholar.google.com/citations?hl=en&user=Ko1n0rAAAAAJ

Other ways I can learn about you

Faculty page:

https://profiles.ucr.edu/app/home/profile/morganj1

NSF CAREER Award:

https://www.nsf.gov/awardsearch/showAward?AWD_ID=2046093&HistoricalAwards=false

Google Scholar:

https://scholar.google.com/citations?hl=en&user=Ko1n0rAAAAAJ&view_op=list_works

Woefully out of date website (sorry):

https://timelab.engr.ucr.edu/

How much money do you need?

$2500-$8000 4 single gene expression vectors at @ $600/ea = $2400 (plus shipping, handling, etc): $2500 6 double gene expression vectors @ $900/ea = $5400 (plus shipping, handling, etc): $5500 Both studies: $8000 The proposal is split into two rounds, screening of individual gene expression, and then planned development of promising combination expression.

Links to any supporting documents or information

No response.

Estimate your probability of succeeding if you get the amount of money you asked for

99% successful synthesis (this can fail, some sequences are hard to synthesize, but this is rare)

90% successful transgenic human cell creation. We’ve generated dozens of human cell lines with tardigrade transgenes, there is not an obvious reason why these would fail

50% The humanized genes improve human cell stress tolerance (we are excited by our current data, but biology is complicated)