ABSTRACT
Articular cartilage is an important hypovascular tissue structure that, once damaged, does not spontaneously
regenerate and often leads to osteoarthritis. Considerable efforts have been made to establish therapies that
biologically repair damaged articular cartilage, which rely heavily on endogenous or exogenous chondrogenic
stem/progenitor cells (CSPCs). One major drawback of current biological therapies is that fibrocartilage tends to
be regenerated, which shows inferior biomechanical properties compared with the healthy hyaline articular
cartilage. Although a number of therapies have been developed to improve the situation, a reproducible method
to regenerates hyaline cartilage that resists endochondral ossification is yet to be developed. Recently, we have
demonstrated that oral administration of type 1 angiotensin II receptor antagonist, losartan, regenerates mostly
hyaline cartilage after microfracture in rabbits, and concomitantly reduces transforming growth factor-beta 1
(TGF-b1) expression. These results suggest that a proper spatiotemporal suppression of TGF-b1 may be critical
to prevent fibrocartilage formation and allow hyaline cartilage regeneration. However, TGF-b is a chondrogenic
factor for CSPCs, and involved in the maintenance of articular cartilage. Furthermore, pharmacological anti-TGF-
b therapies can cause significant unwanted side effects. Therefore, we hypothesize that effective hyaline
cartilage regeneration without overt side effects may be achieved by a cell therapy that also inhibits TGF-b1
signaling locally as needed. Using the CRISPR/Cas9 technology, Dr. Farshid Guilak (mPI) have reported a novel
approach that reprograms stem cells (called Stem cells Modified for Autonomous Regenerative Therapy or
SMART) to make it possible to deliver anti-inflammatory factor in an auto-regulated, feedback-controlled manner,
and demonstrated its utility for musculoskeletal regenerative medicine. In this proposal, we aim to reprogram
therapeutic cells to be able to suppress TGF-b1 action locally around the cells by inducing TGF-b inhibitor from
them whenever TGF-b1 is present in the environment (i.e., autonomous suppression of fibrotic environment).
We consequently propose to test whether such SMART cells may improve cartilage repair when compared to
conventional cells. For this purpose, we will use muscle-derived stem cells (MDSCs) and mesenchymal stromal
cells (MSCs) to reprogram Decorin (Dcn) as the TGF-b1 inhibitor, and the TGF-b-inducible Smad7 gene as the
site to knock-in Dcn (Dcn-KI), using the CRISPR/Cas9 technology. We have already reprogrammed MDSCs,
and our preliminary in vitro results indicate that Decorin is induced in a time & dose dependent manner after
TGF-b1 exposure, and can suppress the fibrotic cascade. We propose to reprogram MSCs using a similar tactic,
and test whether these SMART cells (Dcn-KI MDSCs, Aim1; Dcn-KI MSCs Aim 2) mitigate the effects of TGF-
b1 autonomously and induce long-term repair of hyaline articular cartilage, when compared with control
unmodified cells. Thus, results of this study will provide a proof-of-concept on the utility of the innovative
autoregulatory gene circuit system for development of effective & safe cellular tools for articular cartilage repair.
