Criteria, Challenges, and Opportunities for Acellularized Allogeneic/Xenogeneic Bone Grafts in Bone Repairing.

As bone grafts become more commonly needed by patients and as donors become scarcer, acellularized bone grafts (ABGs) are becoming more popular for restorative purposes. While autogeneic grafts are reliable as a gold standard, allogeneic and xenogeneic ABGs have been shown to be of particular interest due to the limited availability of autogeneic resources and reduced patient well-being in long-term surgeries. Because of the complete similarity of their structures with native bone, excellent mechanical properties, high biocompatibility, and similarities of biological behaviors (osteoinductive and osteoconductive) with local bones, successful outcomes of allogeneic and xenogeneic ABGs in both in vitro and in vivo research have raised hopes of repairing patients' bone injuries in clinical applications. However, clinical trials have been delayed due to a lack of standardized protocols pertaining to acellularization, cell seeding, maintenance, and diversity of ABG evaluation criteria. This study sought to uncover these factors by exploring the bone structures, ossification properties of ABGs, sources, benefits, and challenges of acellularization approaches (physical, chemical, and enzymatic), cell loading, and type of cells used and effects of each of the above items on the regenerative technologies. To gain a perspective on the repair and commercialization of products before implementing new research activities, this study describes the differences between ABGs created by various techniques and methods applied to them. With a comprehensive understanding of ABG behavior, future research focused on treating bone defects could provide a better way to combine the treatment approaches needed to treat bone defects.

Porcine Lymphotropic Herpesviruses (PLHVs) and Xenotranplantation.

Porcine lymphotropic herpesviruses -1, -2 and -3 (PLHV-1, PLHV-2 and PLHV-3) are gammaherpesviruses which are widespread in pigs. They are closely related to the Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus, both of which cause severe diseases in humans. PLHVs are also related to bovine and ovine gammaherpesviruses, which are apathogenic in the natural host, but cause severe diseases after transmission into other species. Until now, no association between PLHVs and any pig diseases had been described. However, PLHV-1 causes a post-transplantation lymphoproliferative disorder (PTLD) after experimental transplantations in minipigs. This disorder is similar to human PTLD, a serious complication of solid human organ transplantation linked to EBV. Xenotransplantation using pig cells, tissues and organs is under development in order to alleviate the shortage of human transplants. Meanwhile, remarkable survival times of pig xenotransplants in non-human primates have been achieved. In these preclinical trials, another pig herpesvirus, the porcine cytomegalovirus (PCMV), a roseolovirus, was shown to significantly reduce the survival time of pig xenotransplants in baboons and other non-human primates. Although PLHV-1 was found in genetically modified donor pigs used in preclinical xenotransplantation, it was, in contrast to PCMV, not transmitted to the recipient. Nevertheless, it seems important to use PLHV-free donor pigs in order to achieve safe xenotransplantation.

US Food and Drug Administration regulatory approaches for xenotransplantation products and xenografts.

The United States Food and Drug Administration's (FDA) regulatory approach for xenotransplantation products and xenografts encompasses regulatory considerations for biological products, medical devices, drugs, combination products, and genetically altered animals, depending on the product. This communication aims to clarify the regulatory approaches and considerations for animal-derived products, specifically xenotransplantation and xenograft products.

Xenotransplantation in Australia: Development of the regulatory process.

In this commentary, we present a brief history of the development of a national regulatory framework for xenotransplantation clinical research in Australia, including the reasons behind the imposition of a 5-year moratorium in 2005 and its subsequent lifting. We conclude with a summary of current relevant guidelines and standards.

Solid organ xenotransplantation at the interface between research and clinical development: Regulatory aspects.

During the last years, progress has been made in survival and function of pig-to-non-human primate organ xenotransplantation using organs from genetically modified pigs and immunosuppression regimens that are clinically acceptable. This, together with increased insights into a low risk of pig-to-human transmission of porcine endogenous retrovirus, has opened the perspective of starting with first-in-human trials with xenogeneic organs. The regulatory path to clinical development is complex. Unlike an organ from human donors, an organ from pigs, either genetically modified or wild-type pigs, is considered a medicinal product for human use and hence is under regulatory oversight, in the United States by the Food and Drug Administration and in Europe by the national competent authorities of the member states as well as the European Medicines Agency. Related to the status of medicinal product, "(current) good practices" apply in the process of generating a xenogeneic organ through to the transplantation into a patient and life-long follow-up. In addition, guidances for xenotransplantation products and genetically modified organisms do apply as well. This commentary focuses on regulatory aspects of transplantation of organs from genetically modified pigs into humans, with the intention to facilitate the interactions between regulatory agencies and institutions (sponsors) in research and clinical development of these organs, to support the perspective of speeding up the process with a proper entry in clinical application, to fill an unmet medical need in patients with end-stage organ disease.

Xenotransplantation of decellularized pig heart valves-Regulatory aspects in Europe.

BACKGROUND: The lack of human donors for allotransplantation forces the development of other strategies to circumvent the existing organ shortage documented on the waiting lists. Here, xenotransplantation offers a suitable option since the genetic modification of animals has become an established method that allows the generation of animals as donors of cells, tissues, and organs with reduced antigenicity. METHODS: Focus is given on the generation of decellularized matrix scaffolds, for example, for valve transplantation and/or repair, that have the potential being fully assimilated by the recipient as they are no longer a mechanical implant with risk of calcification and related failure. RESULTS: This new class of products is transplants that will be regulated either as medical devices or as cell-based medicinal products, that is, advanced therapy medicinal products, according to the regulations in the European Union. CONCLUSIONS: In this review, we compile relevant regulatory aspects and point out the possibilities of how these products for human use may be regulated in the future.

Regulatory aspects of xenotransplantation in Korea.

BACKGROUND: The xenotransplant clinical trial with human subjects seems to be technically feasible, however, it needs the strict regulatory framework from the domestic as well as the international level to make sure the safety of the human subject and the general public. METHODS: The authors reviewed and introduced the current regulations regarding the xenotransplant clinical trial in Korea focusing on the recently stipulated act (Advanced Regenerative Medicine and Biopharmacology Act, ARMBA) and the role of the related government agencies and health institutions. RESULTS AND CONCLUSION: Korea is ready to conduct the xenotransplant clinical trial with human subjects in the current regulatory framework satisfying the requirements of the international guidance. The responsible governmental agencies would collaborate in control the xenotransplant clinical trial under the ARMBA and other related acts and guidance.

Regulatory changes in China on xenotransplantation and related products.

BACKGROUND: Given the persistence and the worldwide shortage of organs from both the deceased and living donors for clinical transplantation, pig organs or tissues hold immense promises for the patients on the waiting list, and xenotransplantation is deemed as one of the solutions to the organ shortage crisis. Indeed, the emerging gene editing technologies, such as CRISPR/Cas9, have led to tremendous progress in the generation of genetically modified pigs to overcome many barriers associated. METHOD: We presented a description of the xenotransplantation regulations in China and the related products. RESULT: Several groups in China have successfully generated transgenic pigs with the elimination of immune rejection or coagulation-related genes, and both pre-clinical and clinical studies have been reported. However, the pre-clinical evaluation and clinical application of porcine xenotransplantation raises ethical and regulatory considerations. Herein, in this review, we will summarize and discuss the progress in xenotransplantation in China and xenotransplantation-related products from the regulatory perspective. CONCLUSION: There has been remarkable progress in both the genetically modified pigs and pre-clinical studies in China, and China will be the first country to successfully fulfill the xenotransplantation from pig organ to human in the near future.

Systematic Establishment of Robustness and Standards in Patient-Derived Xenograft Experiments and Analysis.

Patient-derived xenografts (PDX) are tumor-in-mouse models for cancer. PDX collections, such as the NCI PDXNet, are powerful resources for preclinical therapeutic testing. However, variations in experimental and analysis procedures have limited interpretability. To determine the robustness of PDX studies, the PDXNet tested temozolomide drug response for three prevalidated PDX models (sensitive, resistant, and intermediate) across four blinded PDX Development and Trial Centers using independently selected standard operating procedures. Each PDTC was able to correctly identify the sensitive, resistant, and intermediate models, and statistical evaluations were concordant across all groups. We also developed and benchmarked optimized PDX informatics pipelines, and these yielded robust assessments across xenograft biological replicates. These studies show that PDX drug responses and sequence results are reproducible across diverse experimental protocols. In addition, we share the range of experimental procedures that maintained robustness, as well as standardized cloud-based workflows for PDX exome-sequencing and RNA-sequencing analyses and for evaluating growth. SIGNIFICANCE: The PDXNet Consortium shows that PDX drug responses and sequencing results are reproducible across diverse experimental protocols, establishing the potential for multisite preclinical studies to translate into clinical trials.

Introduction: The Present Status of Xenotransplantation Research.

There is a well-known worldwide shortage of deceased human donor organs for clinical transplantation. The transplantation of organs from genetically engineered pigs may prove an alternative solution. In the past 5 years, there have been sequential advances that have significantly increased pig graft survival in nonhuman primates. This progress has been associated with (1) the availability of increasingly sophisticated genetically engineered pigs; (2) the introduction of novel immunosuppressive agents, particularly those that block the second T-cell signal (costimulation blockade); (3) a better understanding of the inflammatory response to pig xenografts; and (4) increasing experience in the management of nonhuman primates with pig organ or cell grafts. The range of investigations required in experimental studies has increased. The standard immunologic assays are still carried out, but increasingly investigations aimed toward other pathobiologic barriers (e.g., coagulation dysregulation and inflammation) have become more important in determining injury to the graft.Now that prolonged graft survival, extending to months or even years, is increasingly being obtained, the function of the grafts can be more reliably assessed. If the source pigs are bred and housed under biosecure isolation conditions, and weaned early from the sow, most microorganisms can be eradicated from the herd. The potential risk of porcine endogenous retrovirus (PERV) infection remains unknown, but is probably small. Attention is being directed toward the selection of patients for the first clinical trials of xenotransplantation.

The porcine virome and xenotransplantation.

The composition of the porcine virome includes viruses that infect pig cells, ancient virus-derived elements including endogenous retroviruses inserted in the pig chromosomes, and bacteriophages that infect a broad array of bacteria that inhabit pigs. Viruses infecting pigs, among them viruses also infecting human cells, as well as porcine endogenous retroviruses (PERVs) are of importance when evaluating the virus safety of xenotransplantation. Bacteriophages associated with bacteria mainly in the gut are not relevant in this context. Xenotransplantation using pig cells, tissues or organs is under development in order to alleviate the shortage of human transplants. Here for the first time published data describing the viromes in different pigs and their relevance for the virus safety of xenotransplantation is analysed. In conclusion, the analysis of the porcine virome has resulted in numerous new viruses being described, although their impact on xenotransplantation is unclear. Most importantly, viruses with known or suspected zoonotic potential were often not detected by next generation sequencing, but were revealed by more sensitive methods.

Regulatory aspects of clinical xenotransplantation.

Xenotransplantation attracted interest from regulatory authorities, particularly after the demonstration of pig-to-human transmission of porcine endogenous retrovirus (1996). This added to the risk of a product, resulting in a Guidance of the US Food and Drug Administration (2003). This addresses the full flow chart in product manufacturing, starting with the designated pathogen-free status of the source animal; and special aspects regarding the recipient like informed consent and monitoring for infectious pathogens. Also archiving of records from the donor and recipient, as well as storage of samples is described. The European Medicines Agency issued a Guideline on xenogeneic cell therapy products (2009). Cell-based medicinal products are subject to specific regulations and directives, which apply also to xenogeneic products: the xenotransplant guidances/guidelines are an addition to these regulations. Noteworthy, acellular products like heart valves and decellularized cornea are not considered a cell therapy product, but rather a medical device with its own regulation. WHO issued relevant documents, especially about safety, and the International Xenotransplantation Association published consensus documents, a.o., addressing preclinical efficacy requirements before entering clinical trials. This manuscript presents an overview of the regulatory framework, with special focus on cell therapy products necause these are expected to reach the market first (i.e., pancreatic islets, hepatocytes and cellularized cornea); major illustrations are from the European situation. Albeit being complex, the regulation of xenotransplant products does not form a block in product development, but rather supports the introduction of efficacious and safe products to meet the medical need.

Clinical implementation of islet transplantation: A current assessment.

Beta-cell replacement is the only physiologically relevant alternative to insulin injections in patients with type 1 diabetes (T1D). Pancreas and islet transplantation from deceased organ donors can provide a new beta-cell pool to produce insulin, help blood glucose management, and delay secondary diabetes complications. For children and adolescents with T1D, whole pancreas transplantation is not a viable option because of surgical complications, whereas islet transplantation, even if it is procedurally simpler, must still overcome the burden of immunosuppression to become a routine therapy for children in the future.

Review on porcine endogenous retrovirus detection assays--impact on quality and safety of xenotransplants.

Xenotransplantation of porcine organs, tissues, and cells inherits a risk for xenozoonotic infections. Viable tissues and cells intended for transplantation have to be considered as potentially contaminated non-sterile products. The demands on microbial testing, based on the regulatory requirements, are often challenging due to a restricted shelf life or the complexity of the product itself. In Europe, the regulatory framework for xenogeneic cell therapy is based on the advanced therapy medicinal products (ATMP) regulation (2007), the EMA CHMP Guideline on xenogeneic cell-based medicinal products (2009), as well as the WHO and Council of Europe recommendations. In the USA, FDA guidance for industry (2003) regulates the use of xenotransplants. To comply with the regulations, validated test methods need to be established that reveal the microbial status of a transplant within its given shelf life, complemented by strictly defined action alert limits and supported by breeding in specific pathogen-free (SPF) facilities. In this review, we focus on assays for the detection of the porcine endogenous retroviruses PERV-A/-B/-C, which exhibit highly polymorphic proviral loci in pig genomes. PERVs are transmitted vertically and cannot be completely eliminated by breeding or gene knock out technology. PERVs entail a public health concern that will persist even if no evidence of PERV infection of xenotransplant recipients in vivo has been revealed yet. Nevertheless, infectious risks must be minimized by full assessment of pigs as donors by combining different molecular screening assays for sensitive and specific detection as well as a functional analysis of the infectivity of PERV including an adequate monitoring of recipients.

The International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of xenocorneal transplantation.

To develop an international consensus regarding the appropriate conditions for undertaking clinical trials in xenocorneal transplantation, here we review specific ethical, logistical, scientific, and regulatory issues regarding xenocorneal transplantation, and propose guidelines for conduct of clinical xenocorneal transplantation trials. These proposed guidelines are modeled on the published consensus statement of the International Xenotransplantation Association regarding recommended guidelines for conduct of clinical islet xenotransplantation. It is expected that this initial consensus statement will be revised over time in response to scientific advances in the field, and changes in the regulatory framework based on accumulating clinical experience.

Feasibility of salvaging genetic potential of post-mortem fawns: production of sperm in testis tissue xenografts from immature donor white-tailed deer (Odocoileus virginianus) in recipient mice.

The purpose of this study was to evaluate the long-term outcome of testis tissue xenografting from immature deer. Testis tissue was collected post-mortem from a 2-mo-old white-tailed deer fawn (Odocoileus virginianus) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 7 mice; 8 fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo from grafting for up to 14 mo post-grafting. The retrieved xenografts were evaluated for seminiferous tubular density (per mm(2)) and tubular diameter, as well as for seminiferous tubular morphology and identification of the most advanced germ cell type present in each tubule cross section. Overall, 63% of the grafted testis fragments were recovered as xenografts. Testis tissue xenografts showed a gradual testicular development starting with tubular expansion by 2 mo, presence of spermatocytes by 6 mo post-grafting, round and elongated spermatids by 8 mo, followed by fully-formed sperm by 12 mo post-grafting. The timing of complete spermatogenesis generally corresponded to the reported timing of sexual maturation in white-tailed deer. This study demonstrated, for the first time, that testis tissue xenografting from immature deer donors into recipient mice can successfully result in testicular maturation and development of spermatogenesis in the grafts up to the stage of sperm production. These results may therefore provide a model for salvaging genetic material from immature male white-tailed deer that die before reaching sexual maturity.

Xenografting of testis tissue from bison calf donors into recipient mice as a strategy for salvaging genetic material.

The objective was to evaluate the long-term outcome of testis tissue xenografting from neonatal bison calves as a model for closely related rare or endangered ungulates. Testis tissue was collected postmortem from two newborn bison calves (Bison bison bison) and small fragments of the tissue were grafted under the back skin of immunodeficient recipient mice (n = 15 mice; eight fragments/mouse). Single xenograft samples were removed from representative recipient mice every 2 mo after grafting (for up to 16 mo). The retrieved xenografts were evaluated for seminiferous tubular density, tubular diameter, seminiferous tubular morphology, and identification of the most advanced germ cell type. Overall, 69% of the grafted testis fragments were recovered as xenografts. Xenografts weight increased (P < 0.02) approximately four-fold by 2 mo and 10-fold by 16 mo post-grafting. In testis xenografts, gradual maturational changes were evident, manifested as the first detection of the following at the times specified: seminiferous tubule expansion, 2 mo; spermatocytes, 6 mo; round spermatids, 12 mo; and elongated spermatids, 16 mo. Furthermore, there were differences between the two donor calves regarding the efficiency of spermatogenesis in xenografts. The timing of complete spermatogenesis approximately corresponded to the reported timing of sexual maturation in bison. This study demonstrated, apparently for the first time, that testis tissue xenografting from neonatal bison donors into recipient mice resulted in testicular maturation and complete development of spermatogenesis in the grafts.