Review
New Technologies To Enhance In Vivo Reprogramming for Regenerative Medicine

https://doi.org/10.1016/j.tibtech.2018.11.003Get rights and content

Highlights

CRISPR/Cas9 gene-editing systems have been reengineered as programmable transcription factors that can target nearly any gene sequence. A single effector can modulate the expression levels of multiple genes and include epigenetic modifiers.

Synthetic gene circuits and engineered regulatory sequences can facilitate feedback control of transcription factor expression and degradation, permitting combinatorial control of cell states.

Nanoparticles can penetrate through the cell membrane to efficiently deliver biomolecules without inducing acute inflammation, can be used as optical transcriptional actuators, and can specifically permeabilize target cell membranes to trigger factor uptake.

The microenvironment can trigger alterations in cell metabolism and synergize with biophysical cues to adjust the cellular chromatin state. These can be powerful reprogramming pressures when leveraged with biomaterials and soluble factors.

Cellular identity and state are determined by a collection of molecular components that are specified during development and stabilized thereafter to maintain and protect tissue functions. Alteration of the molecular elements (gene expression program and chromatin state) as a result of disease or age can induce somatic cells to assume different identities or modulate functions. Therapeutic use of this technique, called ‘cellular reprogramming’, is very promising for regenerative medicine, but implementation of reprogramming-based strategies in vivo has been precluded by technological and safety limitations. Recent advances in transcriptional control and improved transmembrane delivery strategies now offer exciting potential to more efficiently reprogram cell fates as well as to control the reprogramming timeline and scale of delivery to improve safety.

Section snippets

Why Is It Difficult To Manipulate Molecular Functions of Cells In Vivo?

Mammalian tissue functions are mediated by cell populations that define their state, size, and interaction with other tissues and the environment. The molecular components that drive the fate of these cells and collective tissue behavior are delineated during development and are preserved thereafter through epigenetic regulatory mechanisms. As a result of disease or aging, these cellular programs become dysregulated and organ systems suffer damage or loss.

Re-establishing or reprogramming [1]

CRISPR/Cas9-Based TFs for In Vivo Reprogramming

The type II clustered regularly interspaced short palindromic repeat (CRISPR) system and the CRISPR-associated Cas9 endonuclease are new technologies derived from the bacterial immune system that target and cleave foreign DNA (such as from viruses and plasmids). This elegant system has been engineered to be composed of a short guide RNA (gRNA or sgRNA) coupled to the Cas9 enzyme, and can identify nearly any sequence in the genome that exhibits complementarity to the sequence of the sgRNA, with

Synthetic Biology Tools Offer Combinatorial Gene Regulation for Smarter Reprogramming

Given the importance of TF control for reprogramming, tools from synthetic biology such as new genetic parts or gene circuits, coupled with mathematical modeling and engineering logic, can open new doors in reprogramming and regenerative medicine. For example, gene circuits [30], which are composed of natural or engineered promoters and well-characterized genes, are inducible systems introduced into cells that receive an input (such as a chemical or TF), process the strength of the interaction,

Nanomaterials Enable Delivery of Large Payloads for In Vivo Reprogramming

Nanoscale objects with high aspect ratios (such as nanowires and nanostraws) have emerged as new vehicles to deliver reprogramming factors into cells because they can penetrate into and through the plasma membrane without rupture [41], providing direct access to the cytosol and nucleus [42]. Decorating biomolecules onto the surface or within the nanomaterials enables straightforward delivery of a uniform and large payload to any number of cells that interface with the materials. Instead of

Engineered Biomaterials Can Enhance In Vivo Reprogramming Pressures

Although intrinsic reprogramming pressures have been the primary tool used to initiate changes in cellular programs, several studies have shown that exposing cells to a different microenvironment can reshape their expression profile [60]. These changes are mediated through alterations in metabolism [61] and synergize with other factors present in the microenvironment, such as cytokines [62], to adjust chromatin state [63] and subsequent cellular fate. Given that biomaterials offer the ability

In Vivo Cellular Reprogramming Enhances Functional Recovery Following Disease or Injury

The technologies described above offer exciting possibilities for improving the therapeutic outcomes of regenerative strategies being explored for diabetes, liver fibrosis, heart failure, and central nervous system (CNS) injuries (Table 1). The high prevalence of diabetes, and substantial transcriptional and chromatin similarity between pancreatic β cells and other acinar and endocrine cells, make the pancreas an attractive target for in vivo reprogramming [79]. In the past, AAVs were used to

Concluding Remarks and Future Perspectives

Reproducibly augmenting cell behavior and fate in vivo has already demonstrated the ability to improve functional recovery from injury (myocardial infarction, ischemia/reperfusion injury, CNS injuries) and ameliorate disease (diabetes, liver fibrosis) in animal models. Clinically translating these therapies to humans, however, will require both the use of models more predictive of human therapeutic outcomes and the continued development of delivery strategies that overcome the limitations of

Acknowledgments

The authors acknowledge support from the 3M Foundation and the University of Michigan. The authors regret that, owing to space and reference number limitations, some important studies could not be included.

Glossary

Adeno-associated virus (AAV)
AAV-based vectors are one of the most commonly used intracellular delivery methods for reprogramming. AAV does not integrate into the host cell genome and does not elicit an immune response; however, infected cells are phagocytosed over time. In addition, because they are non-integrating, ectopic transcription factor (TF) expression declines over time.
Lentivirus (LV)
another common method for delivering reprogramming factors. Lentivirus vectors integrate into the host

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