OA

Background of the Osteoarthritis Pharmaceutical Market

The number of osteoarthritis patients in both China and the United States is rapidly increasing1

The vast market size of chemical drugs for osteoarthritis domestically. 2

Existing medications for osteoarthritis treatment fail to meet clinical demands.

Rhegen is pioneering the development of mRNA therapeutics capable of reversing the course of disease.

Osteoarthritis

Introduction

Osteoarthritis (OA) is the most common form of arthritis. Some people refer to it as degenerative joint disease or "wear and tear" arthritis. It most commonly occurs in the hands, hips, and knees. In OA, the cartilage within the joint begins to break down, and changes occur in the underlying bone. These changes typically progress slowly and worsen over time. OA can cause pain, stiffness, and swelling. In some cases, it can also lead to functional impairment and disability; some people are no longer able to perform daily tasks or work.

How can osteoarthritis be treated?

Among the existing modalities, osteoarthritis cannot be cured, hence physicians typically employ various therapies to alleviate its symptoms, which may include ：

Incorporating physical activity

Physical therapy and muscle strengthening exercises

Weight loss

Medications, including over-the-counter analgesics and prescription drugs

Supportive devices, such as canes or walking sticks

Surgery (typically joint replacement surgery if other treatment options are ineffective)

What have we (Rhegen Biotech) done in the treatment of osteoarthritis?

Currently, the main methods for treating OA primarily focus on improving pain symptoms and joint function, including analgesics, nonsteroidal anti-inflammatory drugs (NSAIDs), opioid analgesics, as well as steroids and intra-articular lubricating agents such as hyaluronic acid. Surgical intervention is typically reserved for end-stage OA patients and is considered a last resort.

Regenerative Osteoarthritis mRNA Therapeutic RH116 is an mRNA-based drug product encoding the FGF18 gene. This gene can be translated into the FGF18 protein within the organism, thereby inducing chondrocyte proliferation and increasing extracellular matrix (ECM) generation, demonstrating potential in promoting cartilage growth and repair.

Mechanism of mRNA Design for High Protein Expression

Enhancing Protein Expression Levels in Chondrocytes.

Improving mRNA Stability.

The optimized sequence demonstrates a significant increase in protein expression levels within chondrocytes.

Selection Criteria for Delivery Systems

Non-LNP delivery reduces the risk of hepatic transfer, achieving prolonged local mRNA expression. Upon validation of target protein knee joint expression, RNA candidate drugs continue to exhibit detectable target protein expression 14 days after a single administration.

Process Development

The process development work often takes place after preliminary research and before formal production. Early-stage research on the RSV project has shown promising feasibility of the candidate sequence in preclinical experiments. Therefore, it is necessary to develop related processes with the aim of efficiency, stability, and cost-effectiveness for future scale-up production. Through small-scale process development and validation, higher yields, purity, and cost-effectiveness can often be achieved.

The OA project adopts the company's mature process platform, coordinating the compatibility between independently designed sequences and developed components. The process flow is optimized, streamlining and simplifying where necessary.

The plasmid template purity reaches >99%, with low endotoxin levels of <0.08 EU/μg.

For the stock solution, double-stranded residue is well below 0.04 μg/mg, with a capping efficiency exceeding 97%.

Large-Scale Production

Stable and efficient process development is a prerequisite for scaling up to large-scale production, while compliance with regulatory requirements for production environments is critical for drug application and market approval. The company strictly adheres to GMP regulations in material management, facility management, environmental control, equipment management, and quality management.

Rigorous production management ensures the integrated production of OA throughout the entire process, adhering to the fundamental principles of drug development: safety, efficacy, and quality control. The autonomous platform capabilities are constructed to meet the stringent requirement of completing a batch production within 30 days.

Quality Management

The quality system for tablets is a management system established by pharmaceutical companies to guide and control drug quality. It manages all factors influencing product quality throughout the entire product lifecycle.

In the production of the OA project, central monitoring and quality research are conducted on plasmids, stock solutions, and intermediate products, along with rigorous release testing. This ensures that the production process and product quality meet standards and are comparable across batches.

0	Analysis items	Analytical method	Specification
1	Appearance	Visual inspection	colorless clear or almost clear solution
2	pH	pH meter	7.0±0.5
3	Visible particles	Visual inspection	Should meet requirement
4	Filled volume	Weighing method	Should meet requirement
5	Osmolarity	Crysoscopic metyhod	280~380 mOsmol/kg
6	Sub-visible particles	Light blockage method	≥10um: NMT 6000 particles per container ≥25um: NMT 600 particles per container
7	identification-mRNA sequence accuracy	Sequencing method	The coding sequence should be the same as the theoretical sequence.
8	identification-mRNA sequence length	Capillary electrophoresis	855 nt±20%
9	identification-mRNA sequence integrity	Agarose gel electrophoresis	No obvious degradation and same position of the band compared with reference product.
10	mRNA content	UV	2.00~3.00mg/ml
11	mRNA purity	Capillary electrophoresis	≥70.0%
12	In vitro bioactivity	Cell transfection + ELISA	Report protein expression
13	In vivo bioactivity	Mice method + ELISA	Report protein expression
14	Sterility	Membrane filtration method	Should meet requirement
15	Bacterial endotoxin	Spectrophotometry	≤10EU/dose
16	Abnormal toxicity	Mice + guinea pig method	Should meet requirement

Non-clinical data

mRNA drugs increase cartilage thickness, improve abnormal activity behavior, alleviate joint pain, halt joint cartilage degradation, and result in lower osteophyte scores compared to the untreated group post-administration.

Preclinical Data

The safety data of mRNA drugs is outstanding.

0	entry name	Conclusion of the report
1	Single-dose toxicity trial	There were no significant effects on rat body weight, and animals showed no signs of acute toxicity or mortality. Additionally, there were no significant effects on Beagle dog parameters such as weight, electrocardiogram, body temperature, ophthalmology, hematology, coagulation, blood biochemistry, electrolytes, or urine routine indices post-administration, and no other relevant toxicity reactions or deaths were observed in animals after dosing.
2	Repeated-dose toxicity trial	During the experiment, no animal deaths or euthanasia due to imminent death were observed. At the end of dosing, pathological changes related to the test substance were observed in the synovial membrane of the dosing site joints, manifested as varying degrees of chronic inflammatory cell infiltration and/or synovial epithelial cell proliferation. At the end of the recovery period, synovial membrane changes at cumulative doses of 5 and 10 mg/kg had completely recovered. There were no significant effects on immune indicators in rats and dogs； After continuous administration for one month, there was no significant change in the content of the target protein in the animals. Following a 4-week withdrawal period, the content of the target protein in the animals was below the quantification limit.
3	Special safety trial	SD rats showed local irritation at the injection site after five consecutive intra-articular injections. Did not induce active systemic allergic reactions in guinea pigs.
4	Genetic toxicity test	The results of the Ames test were determined to be negative. There was no induction of chromosomal aberrations in CHL cells. The results of the chromosomal aberration test were determined to be negative. There was no significant bone marrow suppression observed in SD rats, nor was there a notable increase in the micronucleus rate of polychromatic erythrocytes.
5/th>	Safety pharmacology	Under the conditions of this experiment, RH116 at low, medium, and high doses showed no significant effects on the central nervous system of SD rats.
6	Pharmacokinetics	After a single dose in SD rats, mRNA was distributed in the joint cavity lavage fluid, synovium, and articular cartilage, while the protein was mainly distributed in the joint cavity lavage fluid and synovium, with peak levels occurring later than mRNA. With increasing time, both RH116 and protein content gradually decreased.

Clinical submission

The IND application is a crucial step in the development of mRNA products, ensuring compliance with regulatory requirements and clinical approval. How can we meet the requirements for an IND application for mRNA products? We can assist with the IND application process.

OA