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"Genetic Engineering and Imaging Technologies for Osteoarthritis"

 

 

Speaker: Dr. Alejandro Almarza, Associate Professor at the University of Pittsburgh

Day and Date: Friday, September 27th, 2024

Time: 10:45-11:45AM

Location: HST 101

 

 

 

Abstract:

Genetic Engineering for Osteochondral Regeneration

Towards regeneration of the surface of the temporomandibular joint (TMJ) condyle, we developed the goat as a large animal model to test tissue engineering strategies for the TMJ. Our previous approaches have shown robust matrix deposition; however, bone was not observed to regenerate and cartilage-bone interface was irregular and not organized. Thus, we have decided to explore cell-based approaches to better control the architecture of the cartilage and bone layers. Towards this end, we will employ synthetic biology approaches to guide progenitor cell differentiation into both chondrocytes and osteoblasts, and then create bi-layer constructs with both cell types. Clustered Regularly Spaced Palindromic Repeats (CRISPR) epigenome modifications facilitate targeted, multiplex, long-term regulation of endogenous gene expression without altering the base pairs of the target genes. We hypothesize that Sox9 edited BMSC seeded hydrogels will exhibit increased collagen II and GAG deposition, and increased compression properties when compared to controls. To test this hypothesis, we will demonstrate that CRISPR epigenome editing of Sox9 expression in goat BMSCs drives chondrogenic differentiation. Upregulation of Sox9 expression in BMSCs in pellet culture will be confirmed via qPCR. Chondrogenic phenotype will be evaluated by bulk RNA-seq, biochemical analysis, extracellular matrix characterizatio (ECM), histological and immunohistochemical analysis. 

Genetic Engineering for Osteoarthritis Pain 

Recent technological advancements have led to the development of CRISPR epigenome editing systems that facilitate targeted, multiplex, long term regulation of endogenous gene expression without altering the base pairs of the target genes. Previously, we have demonstrated our ability to utilize CRISPR epigenome editing to modulate dorsal root ganglia (DRG) neurons and intervertebral disc (IVD) cell responses to inflammation. We hypothesize that pain and progression of OA in the TMJ is due to the release of the cytokines Interleukin 1 (IL1), tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL6). A key prediction of this hypothesis is that while the cytokines are produced by inflammatory immune cells, knocking down the receptors for these cytokines in innervating neurons will improve pain, and in cells of the articulating tissues it will slow OA progression. To test this hypothesis, we will deliver CRISPR epigenome editing lentiviral vectors that target Interleukin 1 receptor, type I (IL1R1), tumor necrosis factor receptor 1 (TNFR1), and interleukin 6 cytokine family signal transducer (IL6st) directly to the TMJ will regulate TMJ cartilage and TG sensory neuron inflammatory responses, slowing progression of OA and resulting in long-term pain relief.  

Three-dimensional Innervation Patterns of Joints 

We are pioneering 3D visualization techniques to determine the distribution of nerves in the joint. Specifically, tissue clearing protocols (DISCO and/or PEGASUS) accompanied by retrograde adeno-associated virus (AAV) tracers will allow for a never seen before representation of the innervation of both the TMJ and knee. Furthermore, the use of a two-photon ribbon scanner allows for imaging of the entire rodent joint, which will be a huge step forward in whole-joint histological characterization. Currently osteoarthritis histological measures, such as the OARSI score, depend on choosing “representative” sections of the entire area of damage. These new imaging techniques being developed will circumvent the need for  conventional histological methods where tissue needs to be cut into thin sections and then imaged. Instead, the entire joint can now be visualized, eliminating sampling errors, but also showing the localization of the damage in the joint. Additionally, we will further characterize specific  functional neural subtypes by correlating the retrograde labeled neurons  (from joint to ganglia) to neural function as measured by Patch Clamp  Electrophysiology and single cell RNAseq. This will allow for the first step in  the understanding of the disparity between pain sensation in the TMJ when  compared to the knee. Upon total joint replacement with prosthetics, the  vast majority of knee patients have complete relief of pain symptoms.  However, most TMJ patients receiving a total joint prosthetic only go from  an 8 to about a 4 out of 10 in a visual analog scale. This disparity is the  major reason that NIAMS decided to fund The Restoring Joint Health and  Function to Reduce Pain (RE-JOIN) Consortium, with five teams each with  $7 Million total cost for 3 years, and $3 Million for 2 additional years if  milestones are met, for a total of a $50 Million investment by NIAMS on this  topic.

Personal Bio:
Dr. Alejandro Almarza has focused on tissue engineering and regenerative therapies for more than 15 years, and has developed approaches for both soft and hard musculoskeletal tissues. Dr. Almarza has over 45 published manuscripts, with most focused on the temporomandibular joint (TMJ). Dr. Almarza has secured over $12M in NIH funding as PI including three R01 grants, and an UC2 grant from the NIH HEAL Initiative. 
 
Dr. Almarza’s early work focused on the use of extracellular matrix (ECM) scaffolds for regeneration of the TMJ disc. Toward translation, commercialization efforts have involved discussions with FDA, which led to a 2022 paper using good laboratory practices (GLP) large animal studies for safety of the ECM scaffold manufactured under like-good manufacturing practices (GMP) for any TMJ regenerative therapy. In a parallel translational effort, Dr. Almarza was one of the first to publish on the use of magnesium as a degradable metal in craniofacial applications, leading to two issued U.S. patents.
 
Dr. Almarza developed an approach toward regeneration of the mandibular condyle cartilage of the TMJ. Dr. Almarza was the first to publish a regenerative therapy for the mandibular condylar cartilage in a large animal model, where Dr. Almarza pioneered a new type of defect model as well as the goat as an animal model for TMJ regenerative therapies. Future TMJ large animal studies will likely use ruminant species (goats and sheep). 
 
Dr. Almarza’s newest area of interest is in joint degeneration models to understand the link to pain. Dr.  Almarza adapted an abnormal loading model (bite block) from rodents to rabbits, which allowed the  characterization of the mechanical properties of the joint tissues. This new rabbit model required development of methods to study pain responses and joint kinematics.
 
As an advocate and leader, Dr. Almarza contacted the National Institute of Dental and Craniofacial Research (NIDCR) to communicate the concern of the growing TMJ research field that TMJ research did not have a study section with adequate representation of reviewers with the expertise to appreciate the impact and nuances of the TMJ field. Following written and videoconference communications with NIDCR leadership led by Dr. Almarza, the director of NIDCR agreed to raise these concerns to Center for Scientific Review (CSR), which then closely reviewed the data on TMJ applications (e.g., number of submissions, funding rates). Indeed, the CSR with the NIDCR support agreed that TMJ grants were being reviewed in too many different study sections, and the CSR then designated the Skeletal Biology Structure and Regeneration (SBSR) study section as the new home for TMJ proposals, as reflected on the NIH CSR SBSR website. As a direct result of Dr. Almarza’s advocacy, there is now a dedicated study section at the NIH (SBSR) that includes multiple reviewers with the appropriate expertise to evaluate TMJ proposals, including Dr. Almarza himself serving as a regular member of SBSR.

This event is free and open to the public. Please reach out to the Department of Bioengineering's team (inbioe@lehigh.edu) with any questions or comments.

 

Department/Program: 
Bioengineering
College of Engineering