A modified mouse model of Friedreich's ataxia with conditional Fxn allele homozygosity delays onset of cardiomyopathy.

Friedreich's ataxia (FA) is an autosomal recessive disorder caused by a deficiency in frataxin (FXN), a mitochondrial protein that plays a critical role in the synthesis of iron-sulfur clusters (Fe-S), vital inorganic cofactors necessary for numerous cellular processes. FA is characterized by progressive ataxia and hypertrophic cardiomyopathy, with cardiac dysfunction as the most common cause of mortality in patients. Commonly used cardiac-specific mouse models of FA use the muscle creatine kinase (MCK) promoter to express Cre recombinase in cardiomyocytes and striated muscle cells in mice with one conditional Fxn allele and one floxed-out/null allele. These mice quickly develop cardiomyopathy that becomes fatal by 9-11 wk of age. Here, we generated a cardiac-specific model with floxed Fxn allele homozygosity (MCK-Fxnflox/flox). MCK-Fxnflox/flox mice were phenotypically normal at 9 wk of age, despite no detectable FXN protein expression. Between 13 and 15 wk of age, these mice began to display progressive cardiomyopathy, including decreased ejection fraction and fractional shortening and increased left ventricular mass. MCK-Fxnflox/flox mice began to lose weight around 16 wk of age, characteristically associated with heart failure in other cardiac-specific FA models. By 18 wk of age, MCK-Fxnflox/flox mice displayed elevated markers of Fe-S deficiency, cardiac stress and injury, and cardiac fibrosis. This modified model reproduced important pathophysiological and biochemical features of FA over a longer timescale than previous cardiac-specific mouse models, offering a larger window for studying potential therapeutics.NEW & NOTEWORTHY Previous cardiac-specific frataxin knockout models exhibit rapid and fatal cardiomyopathy by 9 wk of age. This severe phenotype poses challenges for the design and execution of intervention studies. We introduce an alternative cardiac-specific model, MCK-Fxnflox/flox, with increased longevity and delayed onset of all major phenotypes. These phenotypes develop to the same severity as previous models. Thus, this new model provides the same cardiomyopathy-associated mortality with a larger window for potential studies.

An Overview of Nonclinical and Clinical Liver Toxicity Associated With AAV Gene Therapy.

This article reviews the presentation given at the 2023 annual meeting of the Society of Toxicologic Pathology (STP) on liver toxicity observed with adeno-associated viral vector (AAV) gene therapy. After decades as a therapeutic modality largely confined to the academic research environment, gene therapy has emerged in recent years as a rapidly expanding therapeutic approach in the biopharmaceutical industry with AAV as the most commonly used viral vector for gene delivery. This interest in the field of gene therapy by industry has been enhanced by the recent success of approved therapies for curing genetic diseases such as ZOLGENSMA for spinal muscular atrophy and LUXTURNA for Leber congenital amaurosis. However, recently reported clinical and nonclinical toxicities highlight the challenges in safely developing AAV gene therapies that require high dose systemic administration. The presentation reviewed general attributes of AAV as a gene therapy vector, clinical and nonclinical liver toxicity associated with AAV gene therapy and the potential for a multimodal immune suppression strategy that may mitigate toxicities.

Scientific and Regulatory Policy Committee Points to Consider: Nonclinical Research and Development of In Vivo Gene Therapy Products, Emphasizing Adeno-Associated Virus Vectors.

Sequencing of the human genome and numerous advances in molecular techniques have launched the era of genetic medicine. Increasingly precise technologies for genetic modification, manufacturing, and administration of pharmaceutical-grade biologics have proved the viability of in vivo gene therapy (GTx) as a therapeutic modality as shown in several thousand clinical trials and recent approval of several GTx products for treating rare diseases and cancers. In recognition of the rapidly advancing knowledge in this field, the regulatory landscape has evolved considerably to maintain appropriate monitoring of safety concerns associated with this modality. Nonetheless, GTx safety assessment remains complex and is designed on a case-by-case basis that is determined by the disease indication and product attributes. This article describes our current understanding of fundamental biological principles and possible procedures (emphasizing those related to toxicology and toxicologic pathology) needed to support research and development of in vivo GTx products. This article is not intended to provide comprehensive guidance on all GTx modalities but instead provides an overview relevant to in vivo GTx generally by utilizing recombinant adeno-associated virus-based GTx-the most common in vivo GTx platform-to exemplify the main points to be considered in nonclinical research and development of GTx products.

Zucker Lean Rats With Hepatic Steatosis Recapitulate Asymptomatic Metabolic Syndrome and Exhibit Greater Sensitivity to Drug-Induced Liver Injury Compared With Standard Nonclinical Sprague-Dawley Rat Model.

Fatty liver disease is a potential risk factor for drug-induced liver injury (DILI). Despite advances in nonclinical in vitro and in vivo models to assess liver injury during drug development, the pharmaceutical industry is still plagued by idiosyncratic DILI. Here, we tested the hypothesis that certain features of asymptomatic metabolic syndrome (namely hepatic steatosis) increase the risk for DILI in certain phenotypes of the human population. Comparison of the Zucker Lean (ZL) and Zucker Fatty rats fed a high fat diet (HFD) revealed that HFD-fed ZL rats developed mild hepatic steatosis with compensatory hyperinsulinemia without increases in liver enzymes. We then challenged steatotic HFD-fed ZL rats and Sprague-Dawley (SD) rats fed normal chow, a nonclinical model widely used in the pharmaceutical industry, with acetaminophen overdose to induce liver injury. Observations in HFD-fed ZL rats included increased liver injury enzymes and greater incidence and severity of hepatic necrosis compared with similarly treated SD rats. The HFD-fed ZL rats also had disproportionately higher hepatic drug accumulation, which was linked with abnormal hepatocellular efflux transporter distribution. Here, we identify ZL rats with HFD-induced hepatic steatosis as a more sensitive nonclinical in vivo test system for modeling DILI compared with SD rats fed normal chow.

Considerations for Preclinical Safety Assessment of Adeno-Associated Virus Gene Therapy Products.

Progress in understanding the molecular bases of human health and disease in recent decades has flourished making it possible for the field of gene therapy (GT) to offer new possibilities for treating, and even curing, a plethora of medical conditions such as monogenic disorders and metabolic diseases. GT is a therapeutic intervention to genetically alter or modify living cells by means of gene delivery achieved using either viral vectors or nonviral vectors, with adeno-associated virus (AAV) vectors constituting market-share majority. Although GT is conceptually attractive, adverse and even fatal iatrogenic complications have marred the initial enthusiasm of clinical successes. The properties of investigational AAV-based GT may pose safety concerns unique from those of small molecule drugs and other macromolecular biologics, such as ectopic or unregulated expression of the transgene, long-term persistence, and off-target distribution. Herein, we discuss considerations in the design of a comprehensive preclinical safety program for AAV-based GT prior to administration in humans.

Nephron segment specific microRNA biomarkers of pre-clinical drug-induced renal toxicity: Opportunities and challenges.

Drug-induced nephrotoxicity is a common drug development complication for pharmaceutical companies. Sensitive, specific, translatable and non-invasive biomarkers of renal toxicity are urgently needed to diagnose nephron segment specific injury. The currently available gold standard biomarkers for nephrotoxicity are not kidney-specific, lack sensitivity for early detection, and are not suitable for renal damage localization (glomerular vs tubulointerstitial injury). MicroRNAs (miRNAs) are increasingly gaining momentum as promising biomarkers of various organ toxicities, including drug induced renal injury. This is mostly due to their stability in easily accessible biofluids, ease of developing nucleic acids detection compared to protein detection assays, as well as their interspecies translatability. Increasing concordance of miRNA findings by standardizing methodology most suitable for their detection and quantitation, as well as characterization of their expression pattern in a cell type specific manner, will accelerate progress toward validation of these miRNAs as biomarkers in pre-clinical, and clinical settings. This review aims to highlight the current pre-clinical findings surrounding miRNAs as biomarkers in two important segments of the nephron, the glomerulus and tubules.

A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors.

Kinases are principal components of signal transduction pathways and the focus of intense basic and drug discovery research. Irreversible inhibitors that covalently modify non-catalytic cysteines in kinase active sites have emerged as valuable probes and approved drugs. Many protein classes, however, have functional cysteines, and therefore understanding the proteome-wide selectivity of covalent kinase inhibitors is imperative. Here, we accomplish this objective using activity-based protein profiling coupled with quantitative MS to globally map the targets, both specific and nonspecific, of covalent kinase inhibitors in human cells. Many of the specific off-targets represent nonkinase proteins that, notably, have conserved active site cysteines. We define windows of selectivity for covalent kinase inhibitors and show that, when these windows are exceeded, rampant proteome-wide reactivity and kinase target-independent cell death conjointly occur. Our findings, taken together, provide an experimental road map to illuminate opportunities and surmount challenges for the development of covalent kinase inhibitors.

Sequence motifs associated with hepatotoxicity of locked nucleic acid--modified antisense oligonucleotides.

Fully phosphorothioate antisense oligonucleotides (ASOs) with locked nucleic acids (LNAs) improve target affinity, RNase H activation and stability. LNA modified ASOs can cause hepatotoxicity, and this risk is currently not fully understood. In vitro cytotoxicity screens have not been reliable predictors of hepatic toxicity in non-clinical testing; however, mice are considered to be a sensitive test species. To better understand the relationship between nucleotide sequence and hepatotoxicity, a structure-toxicity analysis was performed using results from 2 week repeated-dose-tolerability studies in mice administered LNA-modified ASOs. ASOs targeting human Apolipoprotien C3 (Apoc3), CREB (cAMP Response Element Binding Protein) Regulated Transcription Coactivator 2 (Crtc2) or Glucocorticoid Receptor (GR, NR3C1) were classified based upon the presence or absence of hepatotoxicity in mice. From these data, a random-decision forest-classification model generated from nucleotide sequence descriptors identified two trinucleotide motifs (TCC and TGC) that were present only in hepatotoxic sequences. We found that motif containing sequences were more likely to bind to hepatocellular proteins in vitro and increased P53 and NRF2 stress pathway activity in vivo. These results suggest in silico approaches can be utilized to establish structure-toxicity relationships of LNA-modified ASOs and decrease the likelihood of hepatotoxicity in preclinical testing.

Oral administration of soluble guanylate cyclase agonists to rats results in osteoclastic bone resorption and remodeling with new bone formation in the appendicular and axial skeleton.

Orally administered small molecule agonists of soluble guanylate cyclase (sGC) induced increased numbers of osteoclasts, multifocal bone resorption, increased porosity, and new bone formation in the appendicular and axial skeleton of Sprague-Dawley rats. Similar histopathological bone changes were observed in both young (7- to 9-week-old) and aged (42- to 46-week-old) rats when dosed by oral gavage with 3 different heme-dependent sGC agonist (sGCa) compounds or 1 structurally distinct heme-independent sGCa compound. In a 7-day time course study in 7- to 9-week-old rats, bone changes were observed as early as 2 to 3 days following once daily compound administration. Bone changes were mostly reversed following a 14-day recovery period, with complete reversal after 35 days. The mechanism responsible for the bone changes was investigated in the thyroparathyroidectomized rat model that creates a low state of bone modeling and remodeling due to deprivation of thyroid hormone, calcitonin (CT), and parathyroid hormone (PTH). The sGCa compounds tested increased both bone resorption and formation, thereby increasing bone remodeling independent of calciotropic hormones PTH and CT. Based on these studies, we conclude that the bone changes in rats were likely caused by increased sGC activity.

Identification of tubular injury microRNA biomarkers in urine: comparison of next-generation sequencing and qPCR-based profiling platforms.

BACKGROUND: MicroRNAs (miRNAs) are small, non-coding RNAs that regulate protein levels post-transcriptionally. miRNAs play important regulatory roles in many cellular processes and have been implicated in several diseases. Recent studies have reported significant levels of miRNAs in a variety of body fluids, raising the possibility that miRNAs could serve as useful biomarkers. Next-generation sequencing (NGS) is increasingly employed in biomedical investigations. Although concordance between this platform and qRT-PCR based assays has been reported in high quality specimens, information is lacking on comparisons in biofluids especially urine. Here we describe the changes in miRNA expression patterns in a rodent model of renal tubular injury (gentamicin). Our aim is to compare RNA sequencing and qPCR based miRNA profiling in urine specimen from control and rats with confirmed tubular injury. RESULTS: Our preliminary examination of the concordance between miRNA-seq and qRT-PCR in urine specimen suggests minimal agreement between platforms probably due to the differences in sensitivity. Our results suggest that although miRNA-seq has superior specificity, it may not detect low abundant miRNAs in urine samples. Specifically, miRNA-seq did not detect some sequences which were identified by qRT-PCR. On the other hand, the qRT-PCR analysis was not able to detect the miRNA isoforms, which made up the majority of miRNA changes detected by NGS. CONCLUSIONS: To our knowledge, this is the first time that miRNA profiling platforms including NGS have been compared in urine specimen. miRNAs identified by both platforms, let-7d, miR-203, and miR-320, may potentially serve as promising novel urinary biomarkers for drug induced renal tubular epithelial injury.

Mechanistic investigations of test article-induced pancreatic toxicity at the endocrine-exocrine interface in the rat.

Pancreatic toxicity commonly affects the endocrine or exocrine pancreas. However, it can also occur at the endocrine-exocrine interface (EEI), where the capillary network of the islet merges with the capillaries of the surrounding acinar tissue, that is, the insulo-acinar portal system. The goal of this article is to describe a novel, test article-induced pancreatic toxicity that originated at the EEI and to summarize investigations into the mechanistic basis of the injury. This injury was initially characterized by light microscopy in 7/14 day-toxicity studies in Sprague-Dawley (Crl: CD®[SD]) rats with undisclosed test articles. Microvascular injury at the interface resulted in peri-islet serum exudation, fibrin deposition, hemorrhage, inflammation, and secondary degeneration/necrosis of surrounding exocrine tissue. More chronic injury presented as islet fibrosis and lobular atrophy. Direct cytotoxicity affecting the capillary endothelium at the EEI was confirmed ultrastructurally on day 4. Endothelial microparticle and blood flow studies further confirmed endothelial involvement. Similar lesions occurred less frequently in 2 other rat strains and not in the mouse, dog, or cynomolgus macaque. In summary, in vivo and investigative study data confirmed primary endothelial cytotoxicity in the pathogenesis of this lesion and suggested that the lesion may be rat/rat strain-specific and of uncertain relevance for human safety risk assessment.

Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs.

Many current gene therapy targets use recombinant adeno-associated virus (AAV). The majority of delivered AAV therapeutics persist as episomes, separate from host DNA, yet some viral DNA can integrate into host DNA in different proportions and at genomic locations. The potential for viral integration leading to oncogenic transformation has led regulatory agencies to require investigation into AAV integration events following gene therapy in preclinical species. In the present study, tissues were collected from cynomolgus monkeys and mice 6 and 8 weeks, respectively, following administration of an AAV vector delivering transgene cargo. We compared three different next-generation sequencing approaches (shearing extension primer tag selection ligation-mediated PCR, targeted enrichment sequencing [TES], and whole-genome sequencing) to contrast the specificity, scope, and frequency of integration detected by each method. All three methods detected dose-dependent insertions with a limited number of hotspots and expanded clones. While the functional outcome was similar for all three methods, TES was the most cost-effective and comprehensive method of detecting viral integration. Our findings aim to inform the direction of molecular efforts to ensure a thorough hazard assessment of AAV viral integration in our preclinical gene therapy studies.

Regulatory Consideration for the Nonclinical Safety Assessment of Gene Therapies.

The nonclinical safety assessments for gene therapies are evolving, leveraging over 20 years of experimental and clinical experience. Despite the growing experience with these therapeutics, there are no approved harmonized global regulatory documents for developing gene therapies with only the ICH (International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use) S12 guidance on nonclinical biodistribution currently under discussion. Several health authorities have issued guidance over the last 15 years on the nonclinical safety aspects for gene therapy products, but many of the recommendations are limited to high-level concepts on nonclinical safety aspects or altogether silent on key topics. Historically, this approach was appropriately vague given our relatively small dataset of nonclinical experience, where a comprehensive and detailed regulatory guidance approach was unlikely to be appropriate to address all scenarios. However, harmonization of key considerations and assumptions can provide a consistent basis for developing the appropriate nonclinical safety development plans for individual programs, reducing uncertainty across regulatory regions and unnecessary animal use. Several key areas of nonclinical safety testing are nearing maturation for a harmonized approach, including species selection, certain aspects of study design, study duration, and unintended genomic integration risks. Furthermore, several emerging topics are unaddressed in current regulatory guidance for gene therapy products, which will become key areas of differentiation for the next generation of therapeutics. These topics include redosing, juvenile/pediatric safety, and reproductive/developmental safety testing, where relevant experience from other modalities can be applied. The rationale and potential study design considerations for these topics will be proposed, acknowledging that certain aspects of gene therapy development are not considered appropriate for harmonization. This article provides an overview of the current nonclinical safety regulatory landscape, summarizes typical nonclinical safety study designs, highlights areas of uncertainty, and discusses emerging topics that warrant consideration. Specific recommendations and perspectives are provided to inform future regulatory discussions and harmonization efforts.

In vivo overexpression of frataxin causes toxicity mediated by iron-sulfur cluster deficiency.

Friedreich's ataxia is a rare disorder resulting from deficiency of frataxin, a mitochondrial protein implicated in the synthesis of iron-sulfur clusters. Preclinical studies in mice have shown that gene therapy is a promising approach to treat individuals with Friedreich's ataxia. However, a recent report provided evidence that AAVrh10-mediated overexpression of frataxin could lead to cardiotoxicity associated with mitochondrial dysfunction. While evaluating an AAV9-based frataxin gene therapy using a chicken ß-actin promoter, we showed that toxic overexpression of frataxin could be reached in mouse liver and heart with doses between 1 × 1013 and 1 × 1014 vg/kg. In a mouse model of cardiac disease, these doses only corrected cardiac dysfunction partially and transiently and led to adverse findings associated with iron-sulfur cluster deficiency in liver. We demonstrated that toxicity required frataxin's primary function by using a frataxin construct bearing the N146K mutation, which impairs binding to the iron-sulfur cluster core complex. At the lowest tested dose, we observed moderate liver toxicity that was accompanied by progressive loss of transgene expression and liver regeneration. Together, our data provide insights into the toxicity of frataxin overexpression that should be considered in the development of a gene therapy approach for Friedreich's ataxia.

Biodistribution and Tolerability of AAV-PHP.B-CBh-SMN1 in Wistar Han Rats and Cynomolgus Macaques Reveal Different Toxicologic Profiles.

Recombinant adeno-associated viruses (AAVs) have emerged as promising vectors for human gene therapy, but some variants have induced severe toxicity in Rhesus monkeys and piglets following high-dose intravenous (IV) administration. To characterize biodistribution, transduction, and toxicity among common preclinical species, an AAV9 neurotropic variant expressing the survival motor neuron 1 (SMN1) transgene (AAV-PHP.B-CBh-SMN1) was administered by IV bolus injection to Wistar Han rats and cynomolgus monkeys at doses of 2 × 1013, 5 × 1013, or 1 × 1014 vg/kg. A dose-dependent degeneration/necrosis of neurons without clinical manifestations occurred in dorsal root ganglia (DRGs) and sympathetic thoracic ganglia in rats, while liver injury was not observed in rats. In monkeys, one male at 5 × 1013 vg/kg was found dead on day 4. Clinical pathology data on days 3 and/or 4 at all doses suggested liver dysfunction and coagulation disorders, which led to study termination. Histologic evaluation of the liver in monkeys showed hepatocyte degeneration and necrosis without inflammatory cell infiltrates or intravascular thrombi, suggesting that hepatocyte injury is a direct effect of the vector following hepatocyte transduction. In situ hybridization demonstrated a dose-dependent expression of SMN1 transgene mRNA in the cytoplasm and DNA in the nucleus of periportal to panlobular hepatocytes, while quantitative polymerase chain reaction confirmed the dose-dependent presence of SMN1 transgene mRNA and DNA in monkeys. Monkeys produced a much greater amount of transgene mRNA compared with rats. In DRGs, neuronal degeneration/necrosis and accompanying findings were observed in monkeys as early as 4 days after test article administration. The present results show sensory neuron toxicity following IV delivery of AAV vectors at high doses with an early onset in Macaca fascicularis and after 1 month in rats, and suggest adding the autonomic system in the watch list for preclinical and clinical studies. Our data also suggest that the rat may be useful for evaluating the potential DRG toxicity of AAV vectors, while acute hepatic toxicity associated with coagulation disorders appears to be highly species-dependent.

Outcomes of the European Federation of Pharmaceutical Industries and Associations Oligonucleotide Working Group Survey on Nonclinical Practices and Regulatory Expectations for Therapeutic Oligonucleotide Safety Assessment.

The Oligonucleotide Working Group of the European Federation of Pharmaceutical Industries and Associations (EFPIA) conducted a survey of companies to understand the trends in nonclinical practices and regulatory expectations for oligonucleotide drug safety assessment. Twenty-two companies of different types, with varying oligonucleotide experience levels in the field, participated. The survey identified key regulatory challenges and areas of perceived health authority (HA) concern regarding nonclinical safety strategies for oligonucleotides, such as the choice of toxicology species, approaches to dose setting in toxicity studies, dose scaling from animals to humans, the implementation (and regulatory acceptability) of lean packages, and methods for dealing with impurities and human-specific off-targets. The perceived oligonucleotide experience of HAs and the relevance of guidance to oligonucleotide development were also assessed. The results showed a general lack of consensus on nonclinical safety assessment approaches being used for this growing class of medicines and highlight the need for continuing collaboration between sponsors and HAs to better define best practices.

Urinary miRNA Biomarkers of Drug-Induced Kidney Injury and Their Site Specificity Within the Nephron.

Drug-induced kidney injury (DIKI) is a major concern in both drug development and clinical practice. There is an unmet need for biomarkers of glomerular damage and more distal renal injury in the loop of Henle and the collecting duct (CD). A cross-laboratory program to identify and characterize urinary microRNA (miRNA) patterns reflecting tissue- or pathology-specific DIKI was conducted. The overall goal was to propose miRNA biomarker candidates for DIKI that could supplement information provided by protein kidney biomarkers in urine. Rats were treated with nephrotoxicants causing injury to distinct nephron segments: the glomerulus, proximal tubule, thick ascending limb (TAL) of the loop of Henle and CD. Meta-analysis identified miR-192-5p as a potential proximal tubule-specific urinary miRNA candidate. This result was supported by data obtained in laser capture microdissection nephron segments showing that miR-192-5p expression was enriched in the proximal tubule. Discriminative miRNAs including miR-221-3p and -222-3p were increased in urine from rats treated with TAL versus proximal tubule toxicants in accordance with their expression localization in the kidney. Urinary miR-210-3p increased up to 40-fold upon treatment with TAL toxicants and was also enriched in laser capture microdissection samples containing TAL and/or CD versus proximal tubule. miR-23a-3p was enriched in the glomerulus and was increased in urine from rats treated with doxorubicin, a glomerular toxicant, but not with toxicants affecting other nephron segments. Taken together these results suggest that urinary miRNA panels sourced from specific nephron regions may be useful to discriminate the pathology of toxicant-induced lesions in the kidney, thereby contributing to DIKI biomarker development needs for industry, clinical, and regulatory use.

Anti-IL5 decreases the number of eosinophils but not the severity of dermatitis in Sharpin-deficient mice.

Sharpin-deficient (Sharpin(cpdm)) mutant mice develop a chronic eosinophilic dermatitis. To determine the efficacy of eosinophil-depletion in chronic inflammation, Sharpin(cpdm) mice were treated with anti-IL5 antibodies. Mice treated with anti-IL5 had a 90% reduction of circulating eosinophils and a 50% decrease in cutaneous eosinophils after 10 days compared with sham-treated littermates. Reducing the number of eosinophils resulted in increased severity of alopecia and erythema and a significant increase in epidermal thickness. Skin homogenates from mice treated with anti-IL5 had decreased mRNA expression of arylsulfatase B (Arsb), diamine oxidase (amiloride-binding protein 1, also called histaminase; Abp1) and Il10, which are mediators that eosinophils may release to quench inflammation. Skin homogenates from mice treated with anti-IL5 also had decreased mRNA expression of Il4, Il5, Ccl11, kit ligand (Kitl) and Tgfa; and increased mRNA expression of Tgfb1, Mmp12 and tenascin C (Tnc). In order to further decrease the accumulation of eosinophils, Sharpin(cpdm) mice were crossed with IL5 null mice. Il5(-/-), Sharpin(cpdm)/Sharpin(cpdm) mice had a 98% reduction of circulating eosinophils and a 95% decrease in cutaneous eosinophils compared with IL5-sufficient Sharpin(cpdm) mice. The severity of the lesions was similar between IL5-sufficient and IL5-deficient mice. Double mutant mice had a significant decrease in Abp1, and a significant increase in Tgfb1, Mmp12 and Tnc mRNA compared with controls. These data indicate that eosinophils are not essential for the development of dermatitis in Sharpin(cpdm) mice and suggest that eosinophils have both pro-inflammatory and anti-inflammatory roles in the skin of these mice.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Nonclinical Studies that Support Viral Vector-Delivered Gene Therapies: An EFPIA Gene Therapy Working Group Perspective.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question.