"Comparison of Outcomes After Autologous Breast Reconstruction: Latissimus Dorsi with Immediate Fat Transfer vs. Abdominally Based Free Flaps".

BACKGROUND: Autologous breast reconstruction confers favorable patient reports of satisfaction and quality of life compared to implant-based reconstruction over a lifetime. The latissimus dorsi with immediate fat transfer (LIFT) is an alternative approach to abdominally based free flaps (Ab-FF), which expands fully autologous reconstruction to non-microsurgeons. This study aims to compare the two procedures concerning their clinical and patient-reported outcomes one year postoperatively. METHODS: We conducted a retrospective review of LIFTs and Ab-FFs performed between March 2017 and August 2022. The primary outcomes were postoperative complications, reoperations, and longitudinal BREAST-Q scores. BREAST-Q modules included Satisfaction with Breasts, Abdomen, Back, Psychosocial Well-being, Physical Well-being: Chest, Abdomen, Back, and Sexual Well-being. RESULTS: Of the 281 included patients (408 breasts), 211 received Ab-FF, and 70 received LIFT. One-year follow-up (median [IQR]: 12 [12] months) demonstrated that Ab-FF independently predicted dehiscence, reoperation procedures, and revisional surgery. LIFT independently increased the odds of seroma. In addition, obesity predicted dehiscence, while bilateral reconstructions predicted revisional fat grafting. BREAST-Q scores fluctuated over time but were similar across all measured domains by one year postoperatively. CONCLUSIONS: Although Ab-FF is the gold-standard approach for fully autologous reconstruction, LIFT procedures may be associated with a less complicated postoperative course while eliciting similar patient-reported outcomes. LIFT may subsequently be preferred to limit postoperative complications, particularly in patients with obesity. LIFT can also be utilized by more plastic surgeons who are either not microsurgically trained or do not have an environment that fosters Ab-FF.

Free Tissue Transfer to the Lower Extremity in the Setting of Thrombocytosis and the Role of Antiplatelet Therapy: A Propensity Score-Matched Analysis.

BACKGROUND: Free tissue transfer (FTT) provides a versatile method to achieve successful lower limb salvage. Thrombocytosis in patients undergoing lower extremity (LE) FTT is associated with increased risk of complications. The aims of this study were to assess the feasibility of performing LE FTT in patients with preoperative thrombocytosis, and whether antiplatelet (AP) therapy on the day of surgery (DOS) affects outcomes. METHODS: A retrospective review of thrombocytotic patients who underwent LE FTT between 2011 and 2022 was performed. Patients were stratified into groups based on the receipt of AP therapy on the DOS. Patients were propensity score matched for comorbidity burden and postoperative risk stratification. Outcomes of interest included perioperative transfusion requirements, postoperative flap-related complications, rates of flap success, limb salvage, and ambulatory status. RESULTS: Of the 279 patients who underwent LE FTT, 65 (23.3%) were found to have preoperative thrombocytosis. Fifty-three patients remained following propensity score matching; of which, 32 (60.4%) received AP therapy on the DOS and 21 (39.6%) did not. Overall flap success rate was 96.2% (n = 51). The likelihoods of thrombosis and hematoma development were similar between cohorts (p = 0.949 and 0.574, respectively). Receipt of DOS AP therapy was associated an additional 2.77 units and 990.10 mL of transfused blood (p = 0.020 and 0.018, respectively). At a mean follow-up of 20.7 months, overall limb salvage and ambulatory rates were 81.1% (n = 43) and 79.2% (n = 42), respectively, with no differences between cohorts. CONCLUSION: Preoperative thrombocytosis is not an absolute contraindication to LE FTT. DOS AP therapy may be protective in comorbid patients with elevated platelet counts but must be weighed against possible short-term bleeding as suggested by significant increases in postoperative transfusion requirements.

Muscle versus Fascia Free Tissue Transfer for Treatment of Chronic Osteomyelitis in the Comorbid Population.

BACKGROUND: In patients with chronic lower extremity (LE) wounds, chronic osteomyelitis confers additional complexity to achieving adequate treatment. Previous reviews demonstrate increased rates of osteomyelitis recurrence in patients who receive muscle flaps compared with fasciocutaneous flaps for LE limb salvage; however, these studies were not limited to atraumatic populations who receive exclusively free flaps. Thus, this study compared rates of recurrence in chronic osteomyelitis patients undergoing LE reconstruction with fasciocutaneous versus muscle free flaps. METHODS: Patients undergoing free tissue transfer (FTT) between July 2011 and July 2021 were retrospectively reviewed. Patients were stratified into fasciocutaneous and muscle free flap groups. Primary outcomes included osteomyelitis recurrence, flap complications, limb salvage, and ambulatory status. RESULTS: Forty-eight patients with pathologic diagnosis of chronic osteomyelitis of the wound bed were identified, of which 58.3% received fasciocutaneous (n = 28) and 41.7% received muscle flaps (n = 20). The most common comorbidities included diabetes mellitus (n = 29, 60.4%), peripheral neuropathy (n = 27, 56.3%) and peripheral vascular disease (n = 24, 50.0%). Methicillin-resistant or methicillin -sensitive Staphylococcus aureus were the most common pathogen in 18.7% (n = 9) of procedures. The majority of patients underwent a median of three debridements followed by negative pressure wound therapy prior to receiving FTT. At a median follow-up of 16.6 months, the limb salvage and ambulatory rates were 79.2 (n = 38) and 83.3% (n = 40), respectively. The overall rate of microsurgical flap success was 93.8% (n = 45). Osteomyelitis recurred in 25% of patients (n = 12) at a median duration of 4.0 months. There were no significant differences in rates of osteomyelitis recurrence, flap complications, limb salvage, ambulation, and mortality. On multivariate analysis, flap composition remained a nonsignificant predictor of osteomyelitis recurrence (odds ratio: 0.975, p = 0.973). CONCLUSION: This study demonstrates that flap composition may not influence recurrence of osteomyelitis following free flap reconstruction of chronic LE wounds, suggesting that optimal flap selection should be based on wound characteristics and patient goals.

Clinical utility of the modified frailty index in predicting adverse outcomes in patients undergoing lower extremity free flap reconstruction.

BACKGROUND: Identifying at-risk patients for complications remains challenging in patients with chronic lower extremity (LE) wounds receiving free tissue transfer (FTT) for limb salvage. The modified-5 frailty index (mFI-5) has been utilized to predict postoperative complications, yet it has not been studied in this population. The aim of this study was to determine the utility of the mFI-5 in predicting adverse postoperative outcomes. METHODS: Patients ≥60 years, who underwent LE FTT reconstruction at a single institution from 2011 to 2022, were retrospectively reviewed. Patient characteristics, mFI-5, and postoperative outcomes were collected. Cohorts were divided by an mFI-5 score of <2 or ≥2. RESULTS: A total of 115 patients were identified, of which 71.3% (n = 82) were male, 64.3% (n = 74) had a mFI-5 score of ≥2, and 35.7% (n = 41) had a score <2. The average age and body mass index were 67.8 years and 28.7 kg/m2 , respectively. The higher mFI-5 cohort had lower baseline albumin levels (3.0 vs. 4.0 g/dL, p = .015) and higher hemoglobin A1c levels (7.4 vs. 5.8%, p < .001). The postoperative length of stay was longer in the higher mFI-5 cohort (18 vs. 13.4 days, p = .003). The overall flap success was 96.5% (n = 111), with no difference between cohorts (p = .129). Postoperative complications were comparable between cohorts (p = .294). At a mean follow-up of 19.8 months, eight patients (7.0%) underwent amputation, and 91.3% (n = 105) were ambulatory. CONCLUSION: High microsurgical success rates can be achieved in comorbid patients with high frailty indexes who undergo FTT for limb salvage. A multidisciplinary team approach may effectively mitigate negative outcomes in elderly, frail patients.

A comparative analysis of fasciocutaneous versus muscle free flaps in peripheral vascular disease patients.

OBJECTIVE: Chronic lower extremity (LE) wounds are common in patients with peripheral vascular disease (PVD). Free tissue transfer (FTT) provides healthy soft tissue for wound coverage and additional blood supply to promote wound healing. Given previous studies demonstrate increased complications in LE fasciocutaneous flaps, it was hypothesized that low vascular resistance in muscle flaps may be more advantageous for wound healing in PVD patients. Therefore, this study compared outcomes in PVD patients undergoing LE reconstruction with fasciocutaneous versus muscle free flaps. METHODS: Retrospectively reviewed PVD patients undergoing FTT between 2011 and 2021. Patients were stratified into fasciocutaneous and muscle free flap groups. Primary outcomes included complications, flap success, post-reconstruction vascular interventions, limb salvage, and ambulatory status. RESULTS: One hundred thirteen patients with PVD were identified, of which 60.2% received fasciocutaneous (n = 68) and 39.8% received muscle flaps (n = 45). Forty-two patients (37.2%) underwent pre-flap endovascular interventions. Flap success rate was 98.2% (n = 111). Overall complication rate was 41.2% following fasciocutaneous flaps compared to 24.4% in muscle flaps (p = 0.067). Fasciocutaneous flaps had higher odds of ulceration requiring repeat angiogram within 1 year of reconstruction compared to muscle flaps (OR 3.4, 95% CI: 1.07-10.95, p = 0.047), and higher odds of requiring repeat angiogram overall (OR 3.4, 95% CI: 1.07-10.95, p = 0.047). No difference in requiring procedures in the operated limb within 1 year was observed (p = 0.155). At mean follow-up, there was no difference in limb salvage, ambulatory, and mortality rate between groups. CONCLUSION: This study demonstrates that fasciocutaneous flaps had higher postoperative complication rates and more commonly required repeat arteriograms following LE FTT reconstruction due to recurrent ulcerations, suggesting greater utility of muscle flaps for FTT reconstruction in PVD patients.

Long-Term Outcomes in Patients With Peripheral Arterial Disease Who Undergo Free Flap Reconstruction for Chronic Lower Extremity Wounds.

BACKGROUND: Free tissue transfer (FTT) is critical for limb salvage of chronic lower extremity (LE) wounds. In patients with peripheral arterial disease (PAD), FTT LE reconstruction can be challenging due to limited vessel selection for anastomosis. The study aims to evaluate our surgical and functional outcomes after FTT to LE in patients with PAD. METHODS: A retrospective review identified patients who underwent LE free flap reconstruction between 2011 and 2021. All patients underwent preoperative arteriogram and subsequent FTT. Patients were classified into PAD or non-PAD cohorts, based on the presence of LE arterial stenoses or occlusions identified on arteriogram. Primary outcomes included complications, flap success, need for post-FTT vascular reintervention, limb salvage, and ambulatory status. RESULTS: A total of 253 patients underwent FTT to LE, with 84 patients (33.2%) in the PAD cohort. Patients with PAD had a higher prevalence of diabetes (83.3% vs 39.1%, P < 0.001) and end-stage renal disease (8.3% vs 2.4%, P = 0.028). Osteomyelitis was more common in the PAD group (73.8% vs 55.0%, P = 0.004). Free tissue transfer donor sites and flap composition were similar between cohorts. At a mean follow-up of 21.1 months, limb salvage rates were similar between non-PAD and PAD cohorts (90.5% vs 84.5%, P = 0.158), with no significant differences in ambulatory status or mortality. Higher complication rates occurred in the PAD cohort (38.1% vs 20.7%, P = 0.003), of which partial flap necrosis was more prevalent in the PAD group (6.0% vs 0.6%, P = 0.016). There was no difference in flap success rates between groups (P = 0.430). More postflap angiograms were performed in the PAD group (29.8% vs 7.1%, P < 0.001), with repeat percutaneous endovascular intervention performed in 68.0% of the PAD group versus 33.3% of the non-PAD group (P < 0.001). CONCLUSIONS: This is the largest study to demonstrate excellent long-term limb salvage outcomes in patients with PAD who undergo FTT to LE. Percutaneous endovascular intervention and FTT are effective methods to achieve limb salvage in vasculopathic patients with chronic LE wounds.

COVID-19 Vaccination Status and Capsular Contracture Following Prosthetic Breast Reconstruction: A Retrospective, Multicenter Nested Case-Control Study.

BACKGROUND: Capsular contracture (CC) is a common long-term complication following prosthetic-based breast reconstruction (PBBR). Seven cases of CC following mRNA vaccination for coronavirus 2019 (COVID-19) are reported in the literature. OBJECTIVES: The aim of this study was to determine whether receiving the COVID-19 vaccine was associated with CC development following PBBR. METHODS: A retrospective, multicenter nested case-control study was performed from January 2014 to July 2022 of adult female patients who underwent PBBR with acellular dermal matrix placement. Cases of CC were selected if no adjuvant radiation was received and they presented for follow-up between December 2020 and July 2022. Controls included patients who met inclusion criteria but who did not experience CC in either breast. Patient demographics, breast cancer characteristics, reconstructive surgery details, postoperative complications, and COVID-19 exposure details were analyzed and correlated with CC development. RESULTS: Of a total of 230 patients (393 breasts) who received PBBR, 85 patients (135 breasts) met inclusion criteria, of whom 12 patients (19 breasts) developed CC and 73 patients (116 breasts) did not. At the time of median follow-up of 18.1 months (n = 85; interquartile range, 12.2-33.6 months), no statistically significant differences were observed between the short- or long-term complications in cases or controls. There were no significant differences in COVID-19 vaccination status, number of vaccine doses, or vaccination type between cases and controls. Vaccination status was not associated with greater odds of CC development (odds ratio, 1.44; 95% CI, 0.42-5.37; P > .05). CONCLUSIONS: Direct association between CC and COVID-19 vaccination is difficult to prove. Given the known risk of severe COVID-19 infection among immunocompromised patients, those with breast cancer who undergo PBBR should be properly counseled on the benefits and risks of vaccination.

Comparison of Charlson Comorbidity Index and Modified 5-Factor Frailty Index as Clinical Risk Stratification Tools in Predicting Adverse Outcomes in Patients Undergoing Lower Extremity Free-Flap Reconstruction.

BACKGROUND: Free tissue transfer (FTT) reconstruction is associated with a high rate of limb salvage in patients with chronic lower extremity (LE) wounds. Studies have shown perioperative risk stratification tools (e.g., 5-factor modified frailty index [mFI-5] and Charlson comorbidity index [CCI]) to be useful in predicting adverse outcomes; however, no studies have compared them in patients undergoing LE reconstruction. The aim of this study is to compare the utility of mFI-5 and CCI in predicting postoperative morbidity in elderly patients receiving LE FTT reconstruction. METHODS: A retrospective review of patients aged 60 years or older who underwent LE FTT reconstruction from 2011 to 2022 was performed. Comorbidity burden was measured by two validated risk-stratification tools: mFI-5 and CCI. Primary outcomes included prolonged postoperative length of stay (LOS), defined as greater than 75th percentile of the cohort, postoperative complications, and eventual amputation. RESULTS: A total of 115 patients were identified. Median CCI and mFI-5 were 5 (interquartile range [IQR]: 4-6) and 2 (IQR: 1-3), respectively. Average postoperative LOS was 16.4 days. Twenty-nine patients (25.2%) experienced a postoperative complication, and eight patients (7%) required LE amputation at a mean follow-up of 19.8 months. Overall, flap success was 96.5% (n = 111), and limb salvage rate was 93% (n = 108). Increased CCI was found to be independently predictive of only eventual amputation (odds ratio: 1.59; p = 0.039), while mFI-5 was not predictive of prolonged postoperative LOS, flap complications, or eventual amputation. CONCLUSION: This is the first study to compare the utility of mFI-5 and CCI in predicting adverse outcomes in elderly patients undergoing LE FTT reconstruction. Our results demonstrate CCI to be a superior predictor of secondary amputation in this patient population and mFI-5 to have limited utility. Further investigation in a prospective multicenter cohort is warranted.

Intensive care unit versus floor admission following lower extremity free flap surgery: Is there a difference in outcomes?

BACKGROUND: Free tissue transfer (FTT) lower limb salvage requires costly multidisciplinary care. Traditionally, patients who undergo FTT reconstruction for lower extremity (LE) wounds were admitted to the intensive care unit (ICU) in the immediate postoperative period for close monitoring. During the COVID-19 pandemic, our practice shifted toward admitting FTT patients to the floor postoperatively instead of the ICU. The purpose of this study is to compare surgical outcomes in patients admitted to the floor versus ICU immediately following LE free flap reconstruction. METHODS: We retrospectively reviewed patients undergoing LE FTT reconstruction from 2011 to 2021. Flap monitoring consisted of an implantable Cook-Swartz Doppler probe for muscle flaps and ViOptix tissue oximetry for fasciocutaneous flaps; clinical exam and hand-held dopplers were not the primary flap monitoring techniques. Patients were divided into two groups depending on whether they went to the ICU or floor postoperatively. To ensure proper comparability between cohorts, we corrected for age, BMI and Charlson Comorbidity Index (CCI) using 1:2 propensity score matching (floor: ICU). Primary outcomes included early postoperative complications, flap takeback and salvage, flap success, and postoperative length of stay (LOS). RESULTS: A total of 252 patients were identified. Forty-five patients (17.9%) were admitted to the floor postoperatively and 207 patients (82.1%) to the ICU. Overall, microsurgical success rate was 97.2%, which was similar for floor and ICU patients. Flap takeback and salvage were similar between cohorts. Average postoperative LOS was significantly shorter in floor patients (15.7 vs. 19.1 days, p = 0.043). CONCLUSION: Our findings suggest that postoperative floor admission does not decrease flap success rates and should be considered in patients who undergo FTT to LE reconstruction and are otherwise stable. In the ongoing era of health care cost containment, microsurgery centers should consider appropriate floor training to allow medically stable free flap patients to avoid an ICU stay.

Intermittent lipid nanoparticle mRNA administration prevents cortical dysmyelination associated with arginase deficiency.

Arginase deficiency is associated with prominent neuromotor features, including spastic diplegia, clonus, and hyperreflexia; intellectual disability and progressive neurological decline are other signs. In a constitutive murine model, we recently described leukodystrophy as a significant component of the central nervous system features of arginase deficiency. In the present studies, we sought to examine if the administration of a lipid nanoparticle carrying human ARG1 mRNA to constitutive knockout mice could prevent abnormalities in myelination associated with arginase deficiency. Imaging of the cingulum, striatum, and cervical segments of the corticospinal tract revealed a drastic reduction of myelinated axons; signs of degenerating axons were also present with thin myelin layers. Lipid nanoparticle/ARG1 mRNA administration resulted in both light and electron microscopic evidence of a dramatic recovery of myelin density compared with age-matched controls; oligodendrocytes were seen to be extending processes to wrap many axons. Abnormally thin myelin layers, when myelination was present, were resolved with intermittent mRNA administration, indicative of not only a greater density of myelinated axons but also an increase in the thickness of the myelin sheath. In conclusion, lipid nanoparticle/ARG1 mRNA administration in arginase deficiency prevents the associated leukodystrophy and restores normal oligodendrocyte function.

Functional and Patient-reported Outcomes following Transmetatarsal Amputation in High-risk Limb Salvage Patients.

Background: Transmetatarsal amputation (TMA) is performed in patients with nonhealing wounds of the forefoot. Compared with below-knee amputations, healing after TMA is less reliable, and often leads to subsequent higher-level amputation. The aim of this study was to evaluate the functional and patient-reported outcomes of TMA. Methods: A retrospective review of patients who underwent TMA from 2013 to 2021 at our limb-salvage center was conducted. Primary outcomes included postoperative complications, secondary proximal lower extremity amputation, ambulatory status, and mortality. Univariate and multivariate analyses were performed to evaluate independent risk factors for higher-level amputation after TMA. Patient-reported outcome measures for functionality and pain were also obtained. Results: A total of 146 patients were identified. TMA success was achieved in 105 patients (72%), and 41 patients (28%) required higher-level amputation (Lisfranc: 31.7%, Chopart: 22.0%, below-knee amputations: 43.9%). There was a higher incidence of postoperative infection in patients who subsequently required proximal amputation (39.0 versus 9.5%, P < 0.001). At mean follow-up duration of 23.2 months (range, 0.7-97.6 months), limb salvage was achieved in 128 patients (87.7%) and 83% of patients (n = 121) were ambulatory. Patient-reported outcomes for functionality corresponded to a mean maximal function of 58.9%. Pain survey revealed that TMA failure patients had a significantly higher pain rating compared with TMA success patients (P = 0.016). Conclusions: TMA healing remains variable, and many patients will eventually require a secondary proximal amputation. Multi-institutional studies are warranted to identify perioperative risk factors for higher-level amputation and to further evaluate patient-reported outcomes.

CRISPR-Mediated Genomic Addition to CPS1 Deficient iPSCs is Insufficient to Restore Nitrogen Homeostasis.

CPS1 deficiency is an inborn error of metabolism caused by loss-of-function mutations in the CPS1 gene, catalyzing the initial reaction of the urea cycle. Deficiency typically leads to toxic levels of plasma ammonia, cerebral edema, coma, and death, with the only curative treatment being liver transplantation; due to limited donor availability and the invasiveness and complications of the procedure, however, alternative therapies are needed. Induced pluripotent stem cells offer an alternative cell source to partial or whole liver grafts that theoretically would not require immune suppression regimens and additionally are amenable to genetic modifications. Here, we genetically modified CPS1 deficient patient-derived stem cells to constitutively express human codon optimized CPS1 from the AAVS1 safe harbor site. While edited stem cells efficiently differentiated to hepatocyte-like cells, they failed to metabolize ammonia more efficiently than their unedited counterparts. This unexpected result appears to have arisen in part due to transgene promoter methylation, and thus transcriptional silencing, in undifferentiated cells, impacting their capacity to restore the complete urea cycle function upon differentiation. As pluripotent stem cell strategies are being expanded widely for potential cell therapies, these results highlight the need for strict quality control and functional analysis to ensure the integrity of cell products.

Nonlinearities between inhibition and T-type calcium channel activity bidirectionally regulate thalamic oscillations.

Absence seizures result from 3 to 5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.

Split AAV-Mediated Gene Therapy Restores Ureagenesis in a Murine Model of Carbamoyl Phosphate Synthetase 1 Deficiency.

The urea cycle enzyme carbamoyl phosphate synthetase 1 (CPS1) catalyzes the initial step of the urea cycle; bi-allelic mutations typically present with hyperammonemia, vomiting, ataxia, lethargy progressing into coma, and death due to brain edema if ineffectively treated. The enzyme deficiency is particularly difficult to treat; early recognition is essential to minimize injury to the brain. Even under optimal conditions, therapeutic interventions are of limited scope and efficacy, with most patients developing long-term neurologic sequelae. One significant encumberment to gene therapeutic development is the size of the CPS1 cDNA, which, at 4.5 kb, nears the packaging capacity of adeno-associated virus (AAV). Herein we developed a split AAV (sAAV)-based approach, packaging the large transgene and its regulatory cassette into two separate vectors, thereby delivering therapeutic CPS1 by a dual vector system with testing in a murine model of the disorder. Cps1-deficient mice treated with sAAVs survive long-term with markedly improved ammonia levels, diminished dysregulation of circulating amino acids, and increased hepatic CPS1 expression and activity. In response to acute ammonia challenging, sAAV-treated female mice rapidly incorporated nitrogen into urea. This study demonstrates the first proof-of-principle that sAAV-mediated therapy is a viable, potentially clinically translatable approach to CPS1 deficiency, a devastating urea cycle disorder.

Hepatic arginase deficiency fosters dysmyelination during postnatal CNS development.

Deficiency of arginase is associated with hyperargininemia, and prominent features include spastic diplegia/tetraplegia, clonus, and hyperreflexia; loss of ambulation, intellectual disability and progressive neurological decline are other signs. To gain greater insight into the unique neuromotor features, we performed gene expression profiling of the motor cortex of a murine model of the disorder. Coexpression network analysis suggested an abnormality with myelination, which was supported by limited existing human data. Utilizing electron microscopy, marked dysmyelination was detected in 2-week-old homozygous Arg1-KO mice. The corticospinal tract was found to be adversely affected, supporting dysmyelination as the cause of the unique neuromotor features and implicating oligodendrocyte impairment in a deficiency of hepatic Arg1. Following neonatal hepatic gene therapy to express Arg1, the subcortical white matter, pyramidal tract, and corticospinal tract all showed a remarkable recovery in terms of myelinated axon density and ultrastructural integrity with active wrapping of axons by nearby oligodendrocyte processes. These findings support the following conclusions: arginase deficiency is a leukodystrophy affecting the brain and spinal cord while sparing the peripheral nervous system, and neonatal AAV hepatic gene therapy can rescue the defects associated with myelinated axons, strongly implicating the functional recovery of oligodendrocytes after restoration of hepatic arginase activity.

Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

A constitutive knockout of murine carbamoyl phosphate synthetase 1 results in death with marked hyperglutaminemia and hyperammonemia.

The enzyme carbamoyl phosphate synthetase 1 (CPS1; EC 6.3.4.16) forms carbamoyl phosphate from bicarbonate, ammonia, and adenosine triphosphate (ATP) and is activated allosterically by N-acetylglutamate. The neonatal presentation of bi-allelic mutations of CPS1 results in hyperammonemia with reduced citrulline and is reported as the most challenging nitrogen metabolism disorder to treat. As therapeutic interventions are limited, patients often develop neurological injury or die from hyperammonemia. Survivors remain vulnerable to nitrogen overload, being at risk for repetitive neurological injury. With transgenic technology, our lab developed a constitutive Cps1 mutant mouse and reports its characterization herein. Within 24 hours of birth, all Cps1 -/- mice developed hyperammonemia and expired. No CPS1 protein by Western blot or immunostaining was detected in livers nor was Cps1 mRNA present. CPS1 enzymatic activity was markedly decreased in knockout livers and reduced in Cps1+/- mice. Plasma analysis found markedly reduced citrulline and arginine and markedly increased glutamine and alanine, both intermolecular carriers of nitrogen, along with elevated ammonia, taurine, and lysine. Derangements in multiple other amino acids were also detected. While hepatic amino acids also demonstrated markedly reduced citrulline, arginine, while decreased, was not statistically significant; alanine and lysine were markedly increased while glutamine was trending towards significance. In conclusion we have determined that this constitutive neonatal mouse model of CPS1 deficiency replicates the neonatal human phenotype and demonstrates the key biochemical features of the disorder. These mice will be integral for addressing the challenges of developing new therapeutic approaches for this, at present, poorly treated disorder.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

Human hepatocyte transplantation corrects the inherited metabolic liver disorder arginase deficiency in mice.

The transplantation, engraftment, and expansion of primary hepatocytes have the potential to be an effective therapy for metabolic disorders of the liver including those of nitrogen metabolism. To date, such methods for the treatment of urea cycle disorders in murine models has only been minimally explored. Arginase deficiency, an inherited disorder of nitrogen metabolism that presents in the first two years of life, has the potential to be treated by such methods. To explore the potential of this approach, we mated the conditional arginase deficient mouse with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations to give a selective advantage to transplanted (normal) human hepatocytes. On day -1, a uroplasminogen-expressing adenoviral vector was administered intravenously followed the next day with the transplantation of 1 × 106 human hepatocytes (or vehicle alone) by intrasplenic injection. As the initial number of administered hepatocytes would be too low to prevent hepatotoxicity-induced mortality, NTBC cycling was performed to allow for hepatocyte expansion and repopulation. While all control mice died, all except one human hepatocyte transplanted mice survived. Four months after hepatocyte transplantation, 2 × 1011 genome copies of AAV-TBG-Cre recombinase was administered IV to disrupt endogenous hepatic arginase expression. While all control mice died within the first month, human hepatocyte transplanted mice did well. Ammonia and amino acids, analyzed in both groups before and after disruption of endogenous arginase expression, while well-controlled in the transplanted group, were markedly abnormal in the controls. Ammonium challenging further demonstrated the durability and functionality of the human repopulated liver. In conclusion, these studies demonstrate that human hepatocyte repopulation in the murine liver can result in effective treatment of arginase deficiency.

Functional Human and Murine Tissue-Engineered Liver Is Generated from Adult Stem/Progenitor Cells.

Liver disease affects large numbers of patients, yet there are limited treatments available to replace absent or ineffective cellular function of this crucial organ. Donor scarcity and the necessity for immunosuppression limit one effective therapy, orthotopic liver transplantation. But in some conditions such as inborn errors of metabolism or transient states of liver insufficiency, patients may be salvaged by providing partial quantities of functional liver tissue. After transplanting multicellular liver organoid units composed of a heterogeneous cellular population that includes adult stem and progenitor cells, both mouse and human tissue-engineered liver (TELi) form in vivo. TELi contains normal liver components such as hepatocytes with albumin expression, CK19-expressing bile ducts and vascular structures with α-smooth muscle actin expression, desmin-expressing stellate cells, and CD31-expressing endothelial cells. At 4 weeks, TELi contains proliferating albumin-expressing cells and identification of ß2-microglobulin-expressing cells demonstrates that the majority of human TELi is composed of transplanted human cells. Human albumin is detected in the host mouse serum, indicating in vivo secretory function. Liquid chromatography/mass spectrometric analysis of mouse serum after debrisoquine administration is followed by a significant increase in the level of the human metabolite, 4-OH-debrisoquine, which supports the metabolic and xenobiotic capability of human TELi in vivo. Implanted TELi grew in a mouse model of inducible liver failure. Stem Cells Translational Medicine 2017;6:238-248.