Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency.

The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against lethal SARS-CoV-2 challenge in mice. This discovery advances saRNA therapeutics by enabling prolonged protein expression at low doses.

Complete substitution with modified nucleotides suppresses the early interferon response and increases the potency of self-amplifying RNA.

Self-amplifying RNA (saRNA) will revolutionize vaccines and in situ therapeutics by enabling protein expression for longer duration at lower doses. However, a major barrier to saRNA efficacy is the potent early interferon response triggered upon cellular entry, resulting in saRNA degradation and translational inhibition. Substitution of mRNA with modified nucleotides (modNTPs), such as N1-methylpseudouridine (N1mΨ), reduce the interferon response and enhance expression levels. Multiple attempts to use modNTPs in saRNA have been unsuccessful, leading to the conclusion that modNTPs are incompatible with saRNA, thus hindering further development. Here, contrary to the common dogma in the field, we identify multiple modNTPs that when incorporated into saRNA at 100% substitution confer immune evasion and enhance expression potency. Transfection efficiency enhances by roughly an order of magnitude in difficult to transfect cell types compared to unmodified saRNA, and interferon production reduces by >8 fold compared to unmodified saRNA in human peripheral blood mononuclear cells (PBMCs). Furthermore, we demonstrate expression of viral antigens in vitro and observe significant protection against lethal challenge with a mouse-adapted SARS-CoV-2 strain in vivo . A modified saRNA vaccine, at 100-fold lower dose than a modified mRNA vaccine, results in a statistically improved performance to unmodified saRNA and statistically equivalent performance to modified mRNA. This discovery considerably broadens the potential scope of self-amplifying RNA, enabling entry into previously impossible cell types, as well as the potential to apply saRNA technology to non-vaccine modalities such as cell therapy and protein replacement.

Minimally invasive, sustained-release relaxin-2 microparticles reverse arthrofibrosis.

Substantial advances in biotherapeutics are distinctly lacking for musculoskeletal diseases. Musculoskeletal diseases are biomechanically complex and localized, highlighting the need for novel therapies capable of addressing these issues. All frontline treatment options for arthrofibrosis, a debilitating musculoskeletal disease, fail to treat the disease etiology-the accumulation of fibrotic tissue within the joint space. For millions of patients each year, the lack of modern and effective treatment options necessitates surgery in an attempt to regain joint range of motion (ROM) and escape prolonged pain. Human relaxin-2 (RLX), an endogenous peptide hormone with antifibrotic and antifibrogenic activity, is a promising biotherapeutic candidate for musculoskeletal fibrosis. However, RLX has previously faltered through multiple clinical programs because of pharmacokinetic barriers. Here, we describe the design and in vitro characterization of a tailored drug delivery system for the sustained release of RLX. Drug-loaded, polymeric microparticles released RLX over a multiweek time frame without altering peptide structure or bioactivity. In vivo, intraarticular administration of microparticles in rats resulted in prolonged, localized concentrations of RLX with reduced systemic drug exposure. Furthermore, a single injection of RLX-loaded microparticles restored joint ROM and architecture in an atraumatic rat model of arthrofibrosis with clinically derived end points. Finally, confirmation of RLX receptor expression, RXFP1, in multiple human tissues relevant to arthrofibrosis suggests the clinical translational potential of RLX when administered in a sustained and targeted manner.

The Prognosis of Arthrofibroses: Prevalence, Clinical Shortcomings, and Future Prospects.

Fibrosis is the dysregulated biosynthesis of connective tissue that results from persistent infection, high serum cholesterol, surgery, trauma, or prolonged joint immobilization. As a disease that impacts connective tissue, it is prevalent across the body and disrupts normal extracellular and tissue organization. Ultimately, fibrosis impairs the tissue structural, mechanical, or biochemical function. This review describes the clinical landscape of joint fibrosis, that is, arthrofibrosis, including the risk factors and causes, as well as current clinical treatments and their shortcomings. Because treating arthrofibrosis remains an unmet clinical challenge, we present several animal models used for exploration of the physiopathology of arthrofibrosis and summarize their use for testing novel treatments. We then discuss therapeutics for the prevention or treatment of arthrofibrosis that are in preclinical development and in ongoing clinical trials. We conclude with recent findings from molecular biological studies of arthrofibroses that shed insight on future areas of research for improved treatments.

Deimmunized Lysostaphin Synergizes with Small-Molecule Chemotherapies and Resensitizes Methicillin-Resistant Staphylococcus aureus to ß-Lactam Antibiotics.

There is an urgent need for novel agents to treat drug-resistant bacterial infections, such as multidrug-resistant Staphylococcus aureus (MRSA). Desirable properties for new antibiotics include high potency, narrow species selectivity, low propensity to elicit new resistance phenotypes, and synergy with standard-of-care (SOC) chemotherapies. Here, we describe analysis of the antibacterial potential exhibited by F12, an innovative anti-MRSA lysin that has been genetically engineered to evade detrimental antidrug immune responses in human patients. F12 possesses high potency and rapid onset of action, it has narrow selectivity against pathogenic staphylococci, and it manifests synergy with numerous SOC antibiotics. Additionally, resistance to F12 and ß-lactam antibiotics appears mutually exclusive, and, importantly, we provide evidence that F12 resensitizes normally resistant MRSA strains to ß-lactams both in vitro and in vivo These results suggest that combinations of F12 and SOC antibiotics are a promising new approach to treating refractory S. aureus infections.

Globally deimmunized lysostaphin evades human immune surveillance and enables highly efficacious repeat dosing.

There is a critical need for novel therapies to treat methicillin-resistant Staphylococcus aureus (MRSA) and other drug-resistant pathogens, and lysins are among the vanguard of innovative antibiotics under development. Unfortunately, lysins' own microbial origins can elicit detrimental antidrug antibodies (ADAs) that undermine efficacy and threaten patient safety. To create an enhanced anti-MRSA lysin, a novel variant of lysostaphin was engineered by T cell epitope deletion. This "deimmunized" lysostaphin dampened human T cell activation, mitigated ADA responses in human HLA transgenic mice, and enabled safe and efficacious repeated dosing during a 6-week longitudinal infection study. Furthermore, the deimmunized lysostaphin evaded established anti-wild-type immunity, thereby providing significant anti-MRSA protection for animals that were immune experienced to the wild-type enzyme. Last, the enzyme synergized with daptomycin to clear a stringent model of MRSA endocarditis. By mitigating T cell-driven antidrug immunity, deimmunized lysostaphin may enable safe, repeated dosing to treat refractory MRSA infections.

Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 ß-lactamase (P99ßL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 109 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99ßL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.