Therapeutic expression of RAS Degrader RRSP in Pancreatic Cancer via Nanocarrier-mediated mRNA delivery.

About one-third of all human cancers encode abnormal RAS proteins locked in a constitutively activated state to drive malignant transformation and uncontrolled tumor growth. Despite progress in development of small molecules for treatment of mutant KRAS cancers, there is a need for a pan-RAS inhibitor that is effective against all RAS isoforms and variants and that avoids drug resistance. We have previously shown that the naturally occurring bacterial enzyme RAS/RAP1-specific endopeptidase (RRSP) is a potent RAS degrader that can be re-engineered as a biologic therapy to induce regression of colorectal, breast, and pancreatic tumors. Here, we have developed a strategy for in vivo expression of this RAS degrader via mRNA delivery using a synthetic nonviral gene delivery platform composed of the poly(ethylene glycol)-b-poly(propylene sulfide) (PEG-b-PPS) block copolymer conjugated to a dendritic cationic peptide (PPDP2). Using this strategy, PPDP2 is shown to deliver mRNA to both human and mouse pancreatic cells resulting in RRSP gene expression, activity, and loss of cell proliferation. Further, pancreatic tumors are reduced with residual tumors lacking detectable RAS and phosphorylated ERK. These data support that mRNA-loaded synthetic nanocarrier delivery of a RAS degrader can interrupt the RAS signaling system within pancreatic cancer cells while avoiding side effects during therapy.

Attaching artificial Achilles and tibialis cranialis tendons to bone using suture anchors in a rabbit model: assessment of outcomes.

Objective: The purpose of this study was to investigate the factors associated with outcomes of attaching artificial tendons to bone using suture anchors for replacement of biological tendons in rabbits. Study Design: Metal suture anchors with braided composite sutures of varying sizes (USP #1, #2, or #5) were used to secure artificial tendons replacing both the Achilles and tibialis cranialis tendons in 12 New Zealand White rabbits. Artificial tendons were implanted either at the time of (immediate replacement, n=8), or four weeks after (delayed replacement, n=4) resection of the biological tendon. Hindlimb radiographs of the rabbits were obtained immediately after surgery and approximately every other week until the study endpoint (16 weeks post-surgery). Results: All suture anchors used for the tibialis cranialis artificial tendons remained secure and did not fail during the study. The suture linkage between the Achilles artificial tendon and anchor failed in 9 of 12 rabbits. In all cases, the mode of failure was suture breakage distant from the knot. Based on radiographic analysis, the mean estimated failure timepoint was 5.3±2.3 weeks post-surgery, with a range of 2-10 weeks. Analysis of variance (ANOVA) tests revealed no significant effect of tendon implantation timing or suture size on either the timing or frequency of suture anchor failure. Conclusion: Based on the mode of failure, suture mechanical properties, and suture anchor design, we suspect that the cause of failure was wear of the suture against the edges of the eyelet in the suture anchor post, which reduced the suture strength below in vivo loads. Suture anchor designs differed for the tibialis cranialis and did not fail during the period of study. Future studies are needed to optimize suture anchor mechanical performance under different loading conditions and suture anchor design features.

Effect of polyester-based artificial tendons on movement biomechanics: A preliminary in vivo study.

Artificial tendons may be valuable clinical devices for replacing damaged or missing biological tendons. In this preliminary study, we quantified the effect of polyester-suture-based artificial tendons on movement biomechanics. New Zealand White rabbits underwent surgical replacement of either the Achilles (n = 2) or tibialis cranialis (TC, n = 2) biological tendons with artificial tendons. Once pre-surgery and weekly from 2 to 6 weeks post-surgery, we quantified hindlimb kinematics and ground contact pressures during the stance phase of hopping gait. Post-surgical movement biomechanics were either consistent or improved over time in both groups. However, the Achilles group had greater overall biomechanical and muscle deficits than the TC group. In the TC group, at 6 weeks post-surgery, foot angles were about 10° greater than those in healthy controls during the first 30 % of stance. At 6 weeks post-surgery, the Achilles group exhibited lesser (i.e., more dorsiflexed) ankle angles (minimum angle = 31.5 ± 9.4°) and vertical ground reaction forces (37.4 ± 2.6 %BW) during stance than those in healthy controls (65.0 ± 11.2° and 50.2 ± 8.3 %BW, respectively). Future studies are needed to quantify long-term biomechanical function with artificial tendons, the effect of artificial tendons on muscle function and structure, and the effect of formal rehabilitation.

Feasibility of Implanting a Foot-Ankle Endoprosthesis within Skin in a Rabbit Model of Transtibial Amputation.

Prosthetic limbs that are completely implanted within skin (i.e., endoprostheses) could permit direct, physical muscle-prosthesis attachment to restore more natural sensorimotor function to people with amputation. The objective of our study was to test, in a rabbit model, the feasibility of replacing the lost foot after hindlimb transtibial amputation by implanting a novel rigid foot-ankle endoprosthesis that is fully covered with skin. We first conducted a pilot, non-survival surgery in two rabbits to determine the maximum size of the skin flap that could be made from the biological foot-ankle. The skin flap size was used to determine the dimensions of the endoprosthesis foot segment. Rigid foot-ankle endoprosthesis prototypes were successfully implanted in three rabbits. The skin incisions healed over a period of approximately 1 month after surgery, with extensive fur regrowth by the pre-defined study endpoint of approximately 2 months post surgery. Upon gross inspection, the skin surrounding the endoprosthesis appeared normal, but a substantial subdermal fibrous capsule had formed around the endoprosthesis. Histology indicated that the structure and thickness of the skin layers (epidermis and dermis) were similar between the operated and non-operated limbs. A layer of subdermal connective tissue representing the fibrous capsule surrounded the endoprosthesis. In the operated limb of one rabbit, the subdermal connective tissue layer was approximately twice as thick as the skin on the medial (skin = 0.43 mm, subdermal = 0.84 mm), ventral (skin = 0.80 mm, subdermal = 1.47 mm), and lateral (skin = 0.76 mm, subdermal = 1.42 mm) aspects of the endoprosthesis. Our results successfully demonstrated the feasibility of implanting a fully skin-covered rigid foot-ankle endoprosthesis to replace the lost tibia-foot segment of the lower limb. Concerns include the fibrotic capsule which could limit the range of motion of jointed endoprostheses. Future studies include testing of endoprosthetics, as well as materials and pharmacologic agents that may suppress fibrous encapsulation.

Rabbit hindlimb kinematics and ground contact kinetics during the stance phase of gait.

Though the rabbit is a common animal model in musculoskeletal research, there are very limited data reported on healthy rabbit biomechanics. Our objective was to quantify the normative hindlimb biomechanics (kinematics and kinetics) of six New Zealand White rabbits (three male, three female) during the stance phase of gait. We measured biomechanics by synchronously recording sagittal plane motion and ground contact pressure using a video camera and pressure-sensitive mat, respectively. Both foot angle (i.e., angle between foot and ground) and ankle angle curves were unimodal. The maximum ankle dorsiflexion angle was 66.4 ± 13.4° (mean ± standard deviation across rabbits) and occurred at 38% stance, while the maximum ankle plantarflexion angle was 137.2 ± 4.8° at toe-off (neutral ankle angle = 90 degrees). Minimum and maximum foot angles were 17.2 ± 6.3° at 10% stance and 123.3 ± 3.6° at toe-off, respectively. The maximum peak plantar pressure and plantar contact area were 21.7 ± 4.6% BW/cm2 and 7.4 ± 0.8 cm2 respectively. The maximum net vertical ground reaction force and vertical impulse, averaged across rabbits, were 44.0 ± 10.6% BW and 10.9 ± 3.7% BW∙s, respectively. Stance duration (0.40 ± 0.15 s) was statistically significantly correlated (p < 0.05) with vertical impulse (Spearman's ρ = 0.76), minimum foot angle (ρ = -0.58), plantar contact length (ρ = 0.52), maximum foot angle (ρ = 0.41), and minimum foot angle (ρ = -0.30). Our study confirmed that rabbits exhibit a digitigrade gait pattern during locomotion. Future studies can reference our data to quantify the extent to which clinical interventions affect rabbit biomechanics.

Proteolytic pan-RAS Cleavage Leads to Tumor Regression in Patient-derived Pancreatic Cancer Xenografts.

The lack of effective RAS inhibition represents a major unmet medical need in the treatment of pancreatic ductal adenocarcinoma (PDAC). Here, we investigate the anticancer activity of RRSP-DTB, an engineered biologic that cleaves the Switch I of all RAS isoforms, in KRAS-mutant PDAC cell lines and patient-derived xenografts (PDX). We first demonstrate that RRSP-DTB effectively engages RAS and impacts downstream ERK signaling in multiple KRAS-mutant PDAC cell lines inhibiting cell proliferation at picomolar concentrations. We next tested RRSP-DTB in immunodeficient mice bearing KRAS-mutant PDAC PDXs. Treatment with RRSP-DTB led to ≥95% tumor regression after 29 days. Residual tumors exhibited disrupted tissue architecture, increased fibrosis and fewer proliferating cells compared with controls. Intratumoral levels of phospho-ERK were also significantly lower, indicating in vivo target engagement. Importantly, tumors that started to regrow without RRSP-DTB shrank when treatment resumed, demonstrating resistance to RRSP-DTB had not developed. Tracking persistence of the toxin activity following intraperitoneal injection showed that RRSP-DTB is active in sera from immunocompetent mice for at least 1 hour, but absent after 16 hours, justifying use of daily dosing. Overall, we report that RRSP-DTB strongly regresses hard-to-treat KRAS-mutant PDX models of pancreatic cancer, warranting further development of this pan-RAS biologic for the management of RAS-addicted tumors.

RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines.

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. To investigate how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

Fully Implanted Prostheses for Musculoskeletal Limb Reconstruction After Amputation: An In Vivo Feasibility Study.

Previous prostheses for replacing a missing limb following amputation must be worn externally on the body. This limits the extent to which prostheses could physically interface with biological tissues, such as muscles, to enhance functional recovery. The objectives of our study were to (1) test the feasibility of implanting a limb prosthesis, or endoprosthesis, entirely within living skin at the distal end of a residual limb, and (2) identify effective surgical and post-surgical care approaches for implanting endoprostheses in a rabbit model of hindlimb amputation. We iteratively designed, fabricated, and implanted unjointed endoprosthesis prototypes in six New Zealand White rabbits following amputation. In the first three rabbits, the skin failed to heal due to ishemia and dehiscence along the sutured incision. The skin of the final three subsequent rabbits successfully healed over the endoprotheses. Factors that contributed to successful outcomes included modifying the surgical incision to preserve vasculature; increasing the radii size on the endoprostheses to reduce skin stress; collecting radiographs pre-surgery to match the bone pin size to the medullary canal size; and ensuring post-operative bandage integrity. These results will support future work to test jointed endoprostheses that can be attached to muscles.