Based on a RoosterBio Webinar Featuring Bioengineering Assistant Professor Tomas Gonzalez-Fernandez, Ph.D.

MSC 2.0

The term “synthetic biology” (aka synbio) has been redefined and refined with small variations since its early-2000s pioneers. ^{1, 2, 3, 4, 5, 6} While this concept continues to gather steam and is featured at international conferences such as synbiobeta-2024, it also helps to be grounded with a definition that’s simple and concise. For this blog’s purposes, it seems that MIT Professor Krystala Prather, PhD advanced the best one. It is…

“…to re-design existing, natural biological systems for useful purposes (which is really what engineering is all about…)”

Humans have been slowly repurposing biology since the end of the last Ice Age with the domestication of once-wild plants and animals. Today, synbio toolsets now enable life science technologies to progress at a far more rapid rate. In the 2010s, we watched this exciting development converge with CAR-T technology. ⁷ When synthetic biology catapulted “T Cell 1.0” beyond its 1^st -gen, T-cells for adoptive transfer could be upgraded with “T Cell 2.0” genetic operating systems to eliminate liquid tumors.

At RoosterBio we’ve coined the term “MSC 2.0” to describe applications with mesenchymal stem/stromal cells (MSCs) that have been engineered via synthetic biology (e.g., artificial gene programs) and/or systems biology (e.g., MSCs in context of a multi-layered tissue or organoid). ⁸ MSCs’ safe administration across hundreds of “MSC 1.0” clinical trials worldwide and foundation in at least ten marketed cell therapies beyond the USA ⁹ beckons this cell type with “system updates” of its own. ¹⁰ Capable of extended population doublings in large bioreactors, MSCs are emerging as the go-to system for industrialized manufacture of cellular building blocks ¹¹ that can be further customized according to indication and administered allogeneically. ^{12, 13}

RoosterBio is thus eager to engage with intrepid investigators who aim to push back the envelope of MSC 2.0. We recently featured our collaborator, Professor David Vorp, in a webinar (2023) related to MSCs’ and MSC-EVs’ (extracellular vesicles) use in tissue-engineered vascular scaffolds. In a similar spirit, we were of course also delighted to welcome Lehigh University Professor Tomas Gonzalez-Fernandez for a webinar in March 2024 titled Harnessing Synthetic Biology Tools to Engineer Smart Cells and Materials for Musculoskeletal Repair.

Dr. Gonzalez-Fernandez’ Research Vison

Figure 1 (above), Assistant Professor Tomas Gonzalez-Fernandez aims to (1) Engineer smart cells and (2) Engineer smart materials using a combination of state-of-the-art molecular and biomaterials science technologies. The ultimate goal is better alternatives to treat unmet clinical needs related to musculoskeletal diseases.

Dr. Gonzalez-Fernandez is an Assistant Professor in Bioengineering at Lehigh University, having previously completed his postdoctoral fellowship at UC Davis with the lab of Professor Kent Leach and having earned his PhD in Mechanical and Manufacturing Engineering at Dublin’s Trinity College. He began the webinar by describing the unmet need of highly prevalent musculoskeletal disorders (MSDs), which are suffered by approximately 1.7 billion people worldwide with conditions such as cartilage injuries, osteoarthritis, or rheumatoid arthritis. These affect an aging global population as well as an increasing number of young persons, and they not only decrease quality of life but also elevate mortality. As an early-career principal investigator, Gonzalez-Fernandez is attracted to MSCs as a template for bioengineering new treatments and cures. “What we are trying to do is to engineer these cells through gene editing. Specifically, we can differentiate them into different lineages and, also, to modulate the injury environment and guide tissue repair after implantation,” he said.

“Uploading” Cellular Operating Systems

How to do this? First, the effort is to ex vivo engineer “smart cells” (i.e., MSCs) with genetic engineering and gene editing with CRISPR tool kits. A complementary next step is to use CRISPRs for in vivo engineering, optimizing these combinatorial molecular parts on “smart materials” in a targeted and spatiotemporally controllable manner. Together, these would be “…for guiding stem cell function in vivo and producing functional tissues that actively instruct the musculoskeletal repair process.”

 One important technology for cell engineering is genome (or “gene”) editing by CRISPR-Cas9, ¹⁴ for which there are recently approved therapies to treat b-thalassemia and sickle cell disease. ¹⁵ Gene editing technology empowers the cell engineer to disrupt, delete, or edit endogenous gene sequences at precise loci in the genome. It can also insert functional new gene systems into a specific location. This new location can be at an oncogenic-inert address, e.g., a “genomic safe harbor” (GSH) such as Rosa26, AAVS1, CCR5, HPRT, or Hipp11. ¹⁶ Alternatively, one could both “knock-out (ko)” a gene (e.g., to achieve an auxotrophic effect to select for modified cells) and “knock-in (ki)” another replacement gene in the same location with transgene flanking recombination arms. Using the CRISPRki, Dr. Gonzalez-Fernandez began a proof of concept with MSCs to target and insert an artificial reporter gene into the GSH.

MSCs are primary cells and therefore preserve fully intact, anti-oncogenic signaling checkpoints. This built-in “alarm system” underscores MSCs’ outstanding value as exemplar raw material for safer cell therapy. Yet it’s also a double-edged sword. With potentially 100s or 1000s of plasmids suddenly bursting into a pristine cell, ¹⁷ chemical transfection is sometimes observed to “stun” them, arresting their growth, or even inducing overt toxicity. MSC transfection is thus often challenging for non-viral methods. Perhaps due to virus’ evolutionary agenda to perpetuate their life cycle in living host cells, some viral vector transduction protocols of MSCs can be extremely efficient. On the other hand, the raw materials for an ex vivo viral gene therapy (such as CAR-T) involve complex chains of custody, and with them, additional regulatory scrutiny. In addition, some clinical experiences with viral-based gene therapies (i.e., using non-MSCs; ex vivo as well as in vivo) have invoked calls for caution. ^{18, 19, 20}

Considering the pros and cons of chemical vs. viral gene transfer methods, Dr. Gonzalez-Fernandez embraced a challenge to use state-of-the-art non-viral reagents for non-viral delivery. His work recently demonstrated the important advantages to employ the “RALA” cell-penetrating peptide (CPP).

RALA is found to be less toxic for MSCs, allowing them to proliferate, which is necessary for a fully functional CRISPRki system. With the demonstration of a successful targeted knock-in nearly complete, future studies with PhD student, Lisette Werba will integrate synthetic gene circuits into specific loci that can leverage environmental, rheostatic controls of therapeutic transgenes delivered by MSCs.

From Smart Cells to Smart Biomaterials

CRISPR-Cas9 is no longer “just” the Nobel prize-winning, star molecule of 2012 that induces targeted double-strand breaks. ¹⁴ In 2024, it’s now like a whole LEGO kit of diverse biochemical activities that can be snapped together for localization to nearly any precise ~20-nucleotide stretch of genomic or chromatin real estate. ²¹ Together with PhD student Josh Graham, the Gonzalez-Fernandez Lab is using a mutationally inactive dCas9 (i.e., “dead” Cas9) fused with a transactivation domain and GFP reporter. This powerful modality can create a whole catalog of bespoke transcription factors via optimized guide RNAs that direct this nucleoprotein complex to promoter regions of target genes, possibly in multiplex.

With CRISPRa, the transfected nucleic acid does not require a plasmid HDR template, but rather mRNA + gRNA. Since mRNA does not need to enter the nucleus to find its cognate ribosomes, mRNA transfection of MSCs can be quite efficient. RALA-mediated delivery or the CRISPRa system yielded high percentages of cells expressing the heterologous reporter, and an initial proof of principle targeting therapeutically relevant promoters showed robust inductions. CRISPRa is inherently safer than CRISPRki because it does not cut the DNA, a lethal event for the cell if left unrepaired. With the right combinations of CRISPRa tools, Dr. Gonzalez-Fernandez proposes to precisely steer the differentiation of MSCs into useful daughter cells like chondrocytes.

Efficient control of MSC differentiation with CRISPR technology could be an important milestone because it could facilitate industrial-scale production of an important cellular raw material for joint repair and/or reconstructive surgeries. It would also serve as a proof of concept to impel broader use of the technology—in other novel directions. And yet, the key word is “efficient.” The correct combinations of genes must be activated in the correct order. That is why Dr. Gonzalez-Fernandez collaborates with Lehigh U. Professor Lifang He of the Department of Computer Science & Engineering to develop a deep learning model. “So, as a proof of concept, we want to identify different genes that we have to activate in the cells to achieve mature chondrocyte expression profiles,” he explained. The model uses differential expression patterns between RNAseq data from MSCs and chondrocytes such that gene regulatory networks can be mimicked.

Before CRISPRa or CRISPRki can be applicable to humans in vivo, it may be imperative to test drive and fine-tune engineered systems of controlled delivery. These tiny 3D-printed scaffolds could eventually be implanted in situ, in proximity to the injury zone. Professor Gonzalez-Fernandez, together with Professor Leslie Chow and PhD Students Josh Graham and Chiebuka Okpara are developing spatiotemporal control systems for localized CRISPR delivery. With ongoing studies to modulate the chemistry of the scaffold, the timing of molecular release can be controlled.

This webinar naturally many inspired good questions, some related to:

• Applicability of RALA vs. Other Delivery Methods
• Gene Editing Efficiency
• Analytical Techniques for Nanoparticle Delivery
• Future Work and Follow-up

To learn how these questions were answered, tune into the recording of this informative webinar. Until next time, RoosterBio is deeply grateful to learn of these exciting updates from the Gonzalez-Fernandez lab, straight from the center of the action. We most certainly would welcome more inspiring investigators, young and old, to present in webinars such as this one!

 

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