CRISPR-Cas9 base editors and their current role in human therapeutics

BackgroundConsistent progress has been made to create more efficient and useful CRISPR-Cas9-based molecular toolsfor genomic modification.MethodsThis review focuses on recent articles that have employed base editors (BEs) for both clinical and research purposes.ResultsCRISPR-Cas9 BEs are a useful system because of their highefficiency and broad applicability to gene correction and disruption. In addition, base editing has beensuggested as a safer approach than other CRISPR-Cas9-based systems, as it limits double-strand breaksduring multiplex gene knockout and does not require a toxic DNA donor molecule for genetic correction.ConclusionAs such, numerous industry and academic groups are currently developing base editing strategies withclinical applications in cancer immunotherapy and gene therapy, which this review will discuss, with a focuson current and future applications of in vivo BE delivery.     

Cell and gene therapy manufacturing capabilities in Australia and New Zealand

Cell and gene therapy products are rapidly being integrated into mainstream medicine. Developing global capability will facilitate broad access to these novel therapeutics. An initial step toward achieving this goal is to understand cell and gene therapy manufacturing capability in each region. We conducted an academic survey in 2018 to assess cell and gene therapy manufacturing capacity in Australia and New Zealand. We examined the following: the number and types of cell therapy manufacturing facilities; the number of projects, parallel processes and clinical trials; the types of products; and the manufacturing and quality staffing levels. It was found that Australia and New Zealand provide diverse facilities for cell therapy manufacturing, infrastructure and capability. Further investment and development will enable both countries to make important decisions to meet the growing need for cell and gene therapy and regenerative medicine in the region.     

Impact of contracted manufacturing organization protocols on operations in an aca demically based Current Good Manufacturing Practice facility

Immunotherapy of cancer and other diseases is often dependent on adoptive transfer to patients of cellular products generated in Current Good Manufacturing Practice (cGMP) facilities. With the availability and approval of various cellular products for therapy, cell production facilities are experiencing unprecedented growth in demand for services. Increasingly, these services involve processing of externally generated cells for transfer to the bedside. The arrival of cells from external manufacturing facilities for processing and eventual infusion of cell therapy products into patients creates a new layer of responsibility and adds to an already demanding list of the existing procedures in academic cGMP facilities. Sponsors introduce their own requirements for the handling of cells that the laboratory must incorporate and follow. The challenges of creating additional access to cleanrooms, writing new standard operating procedures, expanding personnel training, altering pre-existing schedules and incorporating additional monitoring for safety of external products alter the balance of laboratory operations. Adjustments for accommodating externally manufactured products are numerous and varied, as each sponsor has requests that are product-specific. If cells produced by several different external manufacturers are handled by the same facility, the negative impact on the regular activities in this facility may be considerable. Here the authors provide a review of operational challenges that an academic-based laboratory faces and discuss solutions that could ameliorate the difficulties related to an increasing volume of industry-sponsored trials. The solution may be the development under the auspices of the Foundation for Accreditation of Cellular Therapy or the Food and Drug Administration of regulations that will guide the processing of products manufactured by external companies and make these regulations broadly applicable in all cGMP facilities.     

Resource and hardware options for microarray-based experimentation.

DNA microarray technology permits the study of biological systems and processes on a genome-wide scale. Arrays based on cDNA clones, oligonucleotides and genomic clones have been developed for investigations of gene expression, genetic analysis and genomic changes associated with disease. Over the past 3-4 years, microarrays have become more widely available to the research community. This has occurred through increased commercial availability of custom and generic arrays and the development of robotic equipment that has enabled array printing and analysis facilities to be established in academic research institutions. This brief review examines the public and commercial resources, the microarray fabrication and data capture and analysis equipment currently available to the user.     

Spanish Cell Therapy Network (TerCel): 15 years of successful collaborative translational research

In the current article we summarize the 15-year experience of the Spanish Cell Therapy Network (TerCel), a successful collaborative public initiative funded by the Spanish government for the support of nationwide translational research in this important area. Thirty-two research groups organized in three programs devoted to cardiovascular, neurodegenerative and immune-inflammatory diseases, respectively, currently form the network. Each program has three working packages focused on basic science, pre-clinical studies and clinical application. TerCel has contributed during this period to boost the translational research in cell therapy in Spain, setting up a network of Good Manufacturing Practice–certified cell manufacturing facilities– and increasing the number of translational research projects, publications, patents and clinical trials of the participating groups, especially those in collaboration. TerCel pays particular attention to the public-private collaboration, which, for instance, has led to the development of the first allogeneic cell therapy product approved by the European Medicines Agency, Darvadstrocel. The current collaborative work is focused on the development of multicenter phase 2 and 3 trials that could translate these therapies to clinical practice for the benefit of patients.     

A Framework for Managing Core Facilities within the Research Enterprise

Core facilities represent increasingly important operational and strategic components of institutions'' research enterprises, especially in biomolecular science and engineering disciplines. With this realization, many research institutions are placing more attention on effectively managing core facilities within the research enterprise. A framework is presented for organizing the questions, challenges, and opportunities facing core facilities and the academic units and institutions in which they operate. This framework is intended to assist in guiding core facility management discussions in the context of a portfolio of facilities and within the overall institutional research enterprise.     

Bridging the gap: Facilities and technologies for development of early stage therapeutic mAb candidates

Therapeutic monoclonal antibodies (mAbs) currently dominate the biologics marketplace. Development of a new therapeutic mAb candidate is a complex, multistep process and early stages of development typically begin in an academic research environment. Recently, a number of facilities and initiatives have been launched to aid researchers along this difficult path and facilitate progression of the next mAb blockbuster. Complementing this, there has been a renewed interest from the pharmaceutical industry to reconnect with academia in order to boost dwindling pipelines and encourage innovation. In this review, we examine the steps required to take a therapeutic mAb from discovery through early stage preclinical development and toward becoming a feasible clinical candidate. Discussion of the technologies used for mAb discovery, production in mammalian cells and innovations in single-use bioprocessing is included. We also examine regulatory requirements for product quality and characterization that should be considered at the earliest stages of mAb development. We provide details on the facilities available to help researchers and small-biotech build value into early stage product development, and include examples from within our own facility of how technologies are utilized and an analysis of our client base.Key words: monoclonal antibody, preclinical development, biologics, CHO cells, cell culture     

Regulation of advanced therapy medicinal products in Europe and the role of academia

Background aimsAdvanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them.MethodsEuropean academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented.ResultsFacilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS.ConclusionsGMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.     

U.S. National Institutes of Health Core Consolidation–Investing in Greater Efficiency

The U.S. National Institutes of Health (NIH) invests substantial resources in core research facilities (cores) that support research by providing advanced technologies and scientific and technical expertise as a shared resource. In 2010, the NIH issued an initiative to consolidate multiple core facilities into a single, more efficient core. Twenty-six institutions were awarded supplements to consolidate a number of similar core facilities. Although this approach may not work for all core settings, this effort resulted in consolidated cores that were more efficient and of greater benefit to investigators. The improvements in core operations resulted in both increased services and more core users through installation of advanced instrumentation, access to higher levels of management expertise; integration of information management and data systems; and consolidation of billing; purchasing, scheduling, and tracking services. Cost recovery to support core operations also benefitted from the consolidation effort, in some cases severalfold. In conclusion, this program of core consolidation resulted in improvements in the effective operation of core facilities, benefiting both investigators and their supporting institutions.     

Manufacturing mesenchymal stromal cells for clinical applications: A survey of Good Manufacturing Practices at U.S. academic centers

Background aimsMesenchymal stromal cells (MSC) have gained prominence in the field of regenerative medicine due to their excellent safety profile in human patients and recently demonstrated efficacy in late-stage clinical studies. A prerequisite to achieving successful MSC-based therapies is the development of large-scale manufacturing processes that preserve the biological potency of the founder cell population. Because no standardized manufacturing process exists for MSCs, understanding differences in these processes among U.S. academic facilities would allow for better comparison of results obtained in the clinical setting.MethodsWe collected information through a questionnaire sent to U.S. academic centers that produce MSCs under Good Manufacturing Practice conditions.ResultsThe survey provided information on the number and geographic location of academic facilities in the United States and major trends in their manufacturing practices. For example, most facilities employed MSCs enriched from bone marrow by plastic adherence and expanded in media supplemented with pooled human platelet lysate. Sterility testing and product identification via cell surface phenotype analysis were commonly reported practices, whereas initial and working cell plating densities, culture duration, product formulation and the intended use of the MSC product were highly variable among facilities. The survey also revealed that although most facilities assessed product potency, the methods used were limited in scope compared with the broad array of intended clinical applications of the product.ConclusionsSurvey responses reported herein offer insight into the current best practices used to manufacture MSC-based products in the United States and how these practices may affect product quality and potency. The responses also provide a foundation to establish standardized manufacturing platforms.     

Metrics for Success: Strategies for Enabling Core Facility Performance and Assessing Outcomes

Core Facilities are key elements in the research portfolio of academic and private research institutions. Administrators overseeing core facilities (core administrators) require assessment tools for evaluating the need and effectiveness of these facilities at their institutions. This article discusses ways to promote best practices in core facilities as well as ways to evaluate their performance across 8 of the following categories: general management, research and technical staff, financial management, customer base and satisfaction, resource management, communications, institutional impact, and strategic planning. For each category, we provide lessons learned that we believe contribute to the effective and efficient overall management of core facilities. If done well, we believe that encouraging best practices and evaluating performance in core facilities will demonstrate and reinforce the importance of core facilities in the research and educational mission of institutions. It will also increase job satisfaction of those working in core facilities and improve the likelihood of sustainability of both facilities and personnel.     

TCR transgenes and transgene cassettes for TCR gene therapy: status in 2008

The genetic introduction of T cell receptor genes into T cells has been developed over the past decade as a strategy to induce defined antigen-specific T cell immunity. With the potential value of TCR gene therapy well-established in murine models and the feasibility of infusion of TCR-modified autologous T cells shown in a first phase I trial, the next key step will be to transform TCR gene transfer from an experimental technique into a robust clinical strategy. In this review, we discuss the different properties of the TCR transgene and transgene cassette that can strongly affect both the efficacy and the safety of TCR gene transfer. This paper is a focussed research review based on a presentation given at the sixth annual meeting of the Association for Immunotherapy of Cancer (CIMT), held in Mainz, Germany,15–16 May 2008.     

Crowdfunding,stem cell interventions and autism spectrum disorder: comparing campaigns related to an international “stem cell clinic” and US academic medical center

Background aimsStudies examining crowdfunding campaigns for stem cell interventions have typically focused on campaigns seeking funds to send individuals to businesses marketing unlicensed and unproven stem cell products. However, some crowdfunding campaigns identify academic medical centers as destinations for individuals seeking access to stem cell products provided either in clinical studies or on an expanded access basis. This study examines crowdfunding campaigns seeking funds to enable children diagnosed with autism spectrum disorder access to stem cell interventions.MethodsThis study compares and contrasts crowdfunding campaigns, identifying an international stem cell clinic marketing a purported umbilical cord blood-derived stem cell treatment for autism spectrum disorder, with campaigns soliciting donations intended to help children with autism spectrum disorder either participate in clinical studies or obtain expanded access to stem cell products provided at an academic medical center in the US.ResultsCampaigns connected to both sites contained inaccurate claims. However, campaigns identifying the international clinic as the intended destination site made stronger claims about efficacy and were more reliant upon testimonials than campaigns listing the US-based academic medical center as the planned clinical site. Acknowledging these important distinctions, clinical studies and press releases associated with the academic medical center played an important role in lending the perception of credibility to the putative stem cell treatments marketed by the international clinic.ConclusionsThe study's findings emphasize how important it is for researchers at academic medical centers and comparable research facilities to avoid engaging in stem cell hyperbole; highlight the preliminary nature of early clinical studies; ensure that any claims about safety and efficacy are based upon robust and reliable evidence; and promote responsible science communication by exercising restraint when crafting press releases, conducting media interviews and otherwise publicizing clinical research findings.     

CAR-T cell therapy in India requires a paradigm shift in training,education and health care processes

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of some kinds of cancers. Hundreds of companies and academic institutions are collaborating to develop gene-modified cell therapies using novel targets, different cell types, and manufacturing processes of autologous and allogenic cell therapies. The individualized, custom-made autologous CAR-T cell production platform remains a significant limiting factor for its large-scale clinical application. In this respect, the advances in standardization and automation of the process can have considerable impact on cost reduction. Development of off-the-shelf, ready-to-use universal killer cells can enable scaling up. Despite the wide use of this cell therapy in the United States, Europe and China, its development is limited in developing countries in Southeast Asia, Africa and Latin America. In this review, we focus on good manufacturing practices–compliant manufacturing requirements, operational logistics, and regulatory processes that need to be considered for high-quality gene-modified cell therapies from an Indian perspective. We also list the potential strategies to overcome challenges associated with translation to affordability and scalability.     

关于干细胞临床产业化的一些思考

干细胞的基础研究和临床应用是近几年国内外的热点之一。但是因为没有产业化的明确途径,这个领域的产业化发展缓慢,很有可能像基因治疗和肿瘤疫苗的产业化一样无疾而终。本文探讨了干细胞治疗能够产业化之前需要解决的几个问题。从技术层面,我们比较了胚胎干细胞和成体干细胞,自体干细胞和异体干细胞的优缺点和国内外公司采取的一些途径。从政策方面,我们探讨了把干细胞治疗作为一种医疗技术还是一类医药产品的优缺点,比较了美国FDA和国内监管部门的相关政策,也提出了进一步的问题。最后,我们以美国FDA刚刚批准的Provenge为例,对细胞治疗和干细胞治疗的产业化提出了一些希望和想法。     

Bridging the gap

Therapeutic monoclonal antibodies (mAbs) currently dominate the biologics marketplace. Development of a new therapeutic mAb candidate is a complex, multistep process and early stages of development typically begin in an academic research environment. Recently, a number of facilities and initiatives have been launched to aid researchers along this difficult path and facilitate progression of the next mAb blockbuster. Complementing this, there has been a renewed interest from the pharmaceutical industry to reconnect with academia in order to boost dwindling pipelines and encourage innovation. In this review, we examine the steps required to take a therapeutic mAb from discovery through early stage preclinical development and toward becoming a feasible clinical candidate. Discussion of the technologies used for mAb discovery, production in mammalian cells and innovations in single-use bioprocessing is included. We also examine regulatory requirements for product quality and characterization that should be considered at the earliest stages of mAb development. We provide details on the facilities available to help researchers and small-biotech build value into early stage product development, and include examples from within our own facility of how technologies are utilized and an analysis of our client base.     

Challenges for proteomics core facilities

Many analytical techniques have been executed by core facilities established within academic, pharmaceutical and other industrial institutions. The centralization of such facilities ensures a level of expertise and hardware which often cannot be supported by individual laboratories. The establishment of a core facility thus makes the technology available for multiple researchers in the same institution. Often, the services within the core facility are also opened out to researchers from other institutions, frequently with a fee being levied for the service provided. In the 1990s, with the onset of the age of genomics, there was an abundance of DNA analysis facilities, many of which have since disappeared from institutions and are now available through commercial sources. Ten years on, as proteomics was beginning to be utilized by many researchers, this technology found itself an ideal candidate for being placed within a core facility. We discuss what in our view are the daily challenges of proteomics core facilities. We also examine the potential unmet needs of the proteomics core facility that may also be applicable to proteomics laboratories which do not function as core facilities.     

New challenges in radiobiology research with microbeams

There is a continuing interest in the use of microbeam systems designed to deliver ionizing radiation (both photons and particles) with a resolution of a few micrometers or less in biological targets. With more than 30 facilities currently in operation, several new research topics can be explored. In the 9th International Microbeam Workshop held in Darmstadt, Germany, in July 2010, several new ideas and results have been presented, indicating that microbeams will be increasingly important in radiobiology. Subnuclear targeting of single cells for DNA repair studies and microirradiation of 3D or small animal models are among the most promising new research perspectives.     

Toward precision manufacturing of immunogene T-cell therapies

Cancer can be effectively targeted using a patient's own T cells equipped with synthetic receptors, including chimeric antigen receptors (CARs) that redirect and reprogram these lymphocytes to mediate tumor rejection. Over the past two decades, several strategies to manufacture genetically engineered T cells have been proposed, with the goal of generating optimally functional cellular products for adoptive transfer. Based on this work, protocols for manufacturing clinical-grade CAR T cells have been established, but these complex methods have been used to treat only a few hundred individuals. As CAR T-cell therapy progresses into later-phase clinical trials and becomes an option for more patients, a major consideration for academic institutions and industry is developing robust manufacturing processes that will permit scaling-out production of immunogene T-cell therapies in a reproducible and efficient manner. In this review, we will discuss the steps involved in cell processing, the major obstacles surrounding T-cell manufacturing platforms and the approaches for improving cellular product potency. Finally, we will address the challenges of expanding CAR T-cell therapy to a global patient population.     

The persisting challenge of selective and specific proteasome inhibition

Since the discovery of the proteasome and its structure elucidation intensive research programs in academic institutions and pharmaceutical industries led to identification of a wide spectrum of synthetic and natural small proteasomal inhibitors. Activity studies with these small molecules helped to deeply understand the complex biochemical organization and functioning of the proteasome. The new structural and biochemical insights placed the proteasome as an important anti‐cancer drug target, as revealed by the dipeptide boronate proteasome inhibitor, bortezomib, which is currently used for treatment of multiple myeloma. Serious side effects and partial cell resistance against bortezomib demand creation and discovery of new improved generations of more specific and potent proteasomal inhibitors. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.