Robert M Frederickson

Gesicles: Microvesicle “Cookies” for Transient Information Transfer Between Cells

Readers who have spent much time peering at cultured cells through a microscope may have noticed the membranous debris that accumulates in the culture medium over time. We now know that this “debris” actually comprises components of an elaborate intercellular communication system mediated by membranous extracellular organelles collectively called microvesicles. Given their capacity to transmit information between cells, the types, contents, and functions of these microvesicles are being studied for various applications in many fields. Although a number of reports have shown that proteins overexpressed in cells are incorporated into microvesicles derived from them, their potential for directed informational protein delivery is just now being explored. In this issue of Molecular Therapy, Mangeot et al.1 document microvesicle-mediated transfer of two different proteins that are able to temporarily manipulate the phenotype of the recipient cells. The microvesicles were generated by expression of the spike glycoprotein of vesicular stomatitis virus (VSV-G), which stimulates their production. The authors coined the term “gesicles” to describe the modified microvesicles, which represent an important new functional twist in the expanding and diverse armamentarium of molecular information transfer for therapeutic and experimental applications.

Nonclinical Toxicology in Support of Licensure of Gene Therapies

and discussed their relevance to biologic products. Elesperu stated that genotoxicity assays are designed as surrogate assays to detect rare occurrences of genetic damage, and noted that although no single test alone will detect all types of mutagenic effects, a positive result in any one test is considered evidence for genotoxic potential. Elesperu emphasized that these assays are properly used for hazard identification and not risk translation, and that additional testing to evaluate the risk to humans of insertional mutagenesis by gene therapy vectors will need to be developed in support of eventual licensure. Marion Gruber (FDA) presented the purpose and design of reproductive and developmental toxicity studies. The relevant guidelines, ICH S5a, can be found online at http://www.ich.org/pdfICH/s5a.pdf. Reproductive toxicity studies focus on the effects of a product in pregnant animals, to identify potential developmental defects that might result from fetal exposure to the product. The target population for genetic vaccines and gene therapeutics often includes women in their reproductive years, and the label must have a statement describing the potential risk of using the product during pregnancy. Gruber noted that the evaluation of reproductive and developmental risk of gene therapy vectors should be done on a case-by-case basis, taking into account any evidence for insertional mutagenesis by the vector, the particular target patient population, and any potential for the transgene product to induce disease. Rick Irwin (National Institutes of Environmental Health and Safety) detailed the standard rodent carcinogenicity studies as conducted by the National Toxicology Program (http://ntp.niehs.nih.gov), emphasizing that these studies are designed to evaluate the occurrence of both benign and malignant growth. Irwin noted that carcinogenicity studies are both time-consuming and costly, and that they therefore must be carefully planned and implemented, and that the requirements for the testing of gene transfer vectors in rodents should be determined by risks associated with the specific gene transfer agent. Leslie Recio (Merck Research Laboratories) discussed alternative models for carcinogenicity testing that make use of genetically modified animals that have been ‘primed’ for tumor development. Potential advantages of such systems are greater sensitivity that could lead to earlier detection of malignant potential using a smaller number of animals. Richard D. McFarland (FDA) next discussed the timing of nonclinical studies of therapeutic MEETING REPORT

Gene delivery to the nervous system

The National Institute of Neurological Disorders and Stroke (NINDS) sponsored a workshop on gene delivery to the nervous system, which took place on 12–13 November 2007 in Washington, DC. The purpose of the workshop was to convene neuroscientists, molecular virologists/vectorologists, and surgical neurologists to assess the state of the art of gene therapy for neurologic diseases and brain tumors and to address the challenges for advancing promising preclinical studies to the clinic.

A New Era of Innovation for CAR T-cell Therapy

The field of chimeric antigen receptor (CAR) T-cell therapies has entered a new stage of development, and the tremendous promise of this new cancer treatment was evident at the recent CAR-T Summit held in Boston 12–13 November 2015. The meeting was organized by Hanson-Wade, a commercial enterprise specializing in business conferences, including some within the biotechnology and life-sciences sectors. The aim of the conference was to bring together diverse stakeholders to discuss how to facilitate the commercial development of this rapidly evolving field. The larger part of the meeting was dedicated to issues of efficiency, cost, logistics, and quality control of product manufacturing, in addition to regulatory challenges and prospects for reimbursement by health-care payers. Although these issues pose important hurdles to the field, there was collective optimism among the attendees that they would eventually be surmounted.The concept of CAR T-cell therapy dates to the 1980s, when Zelig Eshhar and colleagues engineered and expressed chimeric T-cell receptor (TcR) genes comprising the TcR constant domains fused to the variable domain from an antibody molecule.1xExpression of immunoglobulin-T-cell receptor chimeric molecules as functional receptors with antibody-type specificity. Gross, G, Waks, T, and Eshhar, Z. PNAS. 1989; 86: 10024–10028Crossref | PubMed | Scopus (331)See all References1 The aim was to redirect the specificity of the engineered T cells toward an antigen of choice—such as a tumor-specific antigen—in a manner independent of the major histocompatibility complex. Assuming the selection of an appropriate target antigen restricted to tumor cells, the idea is to thus direct a patients own T cells to express the chimeric receptor and then to reinfuse the cells into the patient to attack and kill the antigen-bearing tumor. In the intervening years, second- and third-generation chimeric receptors have been developed that augment the potency of the therapy.A pertinent question is whether we might be witnessing another rush toward commercialization that might end in disappointment. The biotechnology industry has seen several boom-and-bust cycles with the development of a number of promising platforms—antisense technology, RNA interference, and oncolytic virotherapy being easy examples. What distinguishes CAR T-cell technology from these platforms, however, is the robustness of the early preclinical and clinical data—at least for therapy targeting the B-cell CD19 antigen. Several laboratories and centers have reported success in early trials, at this point primarily for hematological malignancies. The challenge now is establishing and putting into place manufacturing technology that can meet the anticipated demand for the treatment, and in a fashion that is economically sustainable.2xAdoptive cellular therapy: A race to the finish line. June, CH, Riddell, SR, and Schumacher, TN. Sci Trans Med. 2015; 7: 280ps7Crossref | PubMed | Scopus (75)See all References2CAR T-cell therapy would not be the first cell therapy to hit the market. Dendreons Provenge—an autologous cell therapy product for prostate cancer—was approved by the US Food and Drug Administration in 2010. Unfortunately, the expensive therapy afforded only modest benefit, and Dendreon was bankrupt by the end of 2014. Despite the failure of Provenge, Dendrons example shows that cell therapy products can be brought to market, but that cost and efficacy, and the balance between the two, will be important factors determining their success or failure.As noted by Usman Azam, the Global Head of the Cell & Gene Therapies Unit at Novartis Pharmaceuticals, “We have moved on from the era of a “cottage industry” in relation to manufacturing science and now realizing true scalability of therapies like CAR-T. But much more will need to be done to ensure all stakeholders can meet the demand globally and ensure consistent and quality products for our patients.” Indeed, the number of cellular products available for patients should rise with increased automation of what is currently a manual process dependent on highly trained technicians.Other challenges and opportunities remain. Efforts are under way to engineer cells to create allogeneic “off-the-shelf” products, which obviate the need for a personalized therapy. At the December meeting of the American Society of Hematology, a report was recently presented on the first clinical application of “universal” CD19-targeted CAR T cells modified by transcription activator–like effector nucleases to knock out both the endogenous T-cell receptors and CD52, which effectively eliminates the risk of graft-versus-host disease.3xQasim, W, Amrolia, PJ, Samarasinghe, S, Ghorashian, S, Zhan, H, Stafford, S et al. See all References3 The therapy was used on a compassionate basis under UK special-therapy regulations for an infant with refractory, relapsed B-cell acute lymphocytic leukemia. Although the follow-up period is still quite short, the intervention, comprising lymphodepletion and infusion of the universal CAR T cells, has induced molecular remission where all other treatments had failed.Both on-target and off-target recognition of normal tissue can occur with engineered T cells, and adverse events and toxicities have been observed in the clinic. These effects are being mitigated through the development of genetic safety switches and increasing the potency of the cell therapy so as to limit the doses required. Others are adapting the technology for solid tumors and other disease indications (see the Research Highlights in this issue). What is clear is that we can expect a continuing stream of encouraging clinical results that should help drive innovation in the manufacturing process as well as further refinement of the technology itself so that the ultimate aim of bringing this life-saving therapy to patients is realized.

Molecular Therapy Special Issue on Gene Editing Technologies and Applications.

It has been a busy couple of years for the ASGCT publishing program. Last year saw the launch of two additional sibling journals: Molecular Therapy—Methods and Clinical Development in January and Molecular Therapy—Oncolytics in December. These additions to the Molecular Therapy portfolio expand our capacity to publish the best gene and cell therapy studies by capitalizing on the increased pace of translational and commercial development in gene and cell therapy overall and specifically in the burgeoning fields of T-cell therapies and oncolytic virotherapy. Although some might question the need for more journals serving our communities, this is a time of great change for the business of science publishing, and our aim is to both meet the challenges engendered by this change and exploit the opportunities provided by these same challenges.

A Question of Reproducibility

In all science, error precedes the truth, and it is better it should go first than last. —Hugh Walpole Reproducibility is a fundamental tenet of the scientific process, and sharing of both data and methodology in a public forum is key to ensuring reproducibility of scientific findings. However, in recent years there has been increasing concern over the lack of reproducibility of many published scientific findings. This issue has been exacerbated by the rapid expansion of new publishing models, such as open-access journals and preprint servers, not all of which have same requirements with regard to data reproducibility and rigor of the review process. This is not a trivial issue, as irreproducible data can send basic and preclinical researchers down blind alleys, wasting both time and money. This has prompted action by stakeholders in the integrity of the scientific process. Besides scientists, these include funders, academic societies, publishers, and, of course, the public, including patient-advocacy groups. For example, the Prostate Cancer Foundation (PCF) recently announced the Movember Foundation–PCF Scientific Reproducibility Initiative, which aims to accelerate translation of promising discoveries into new tests and treatments for prostate cancer through faster validation of the science underlying new experimental therapies. This past July, the Office of Science and Technology Policy and the National Economic Council asked how the federal government could leverage its role as a significant funder of scientific research to address the reproducibility issue. Publishers and academic societies are also tackling this key issue. Nature Publishing Group (NPG) has taken an active lead in establishing means to fight irreproducibility. NPG introduced measures last year with a view to ensuring good reporting standards and improving the reproducibility of findings published in the pages of its life-science journals. One such measure is requiring completion of a checklist (http://www.nature.com/authors/policies/checklist.pdf). In June, NPG cohosted a meeting with the leadership of the National Institutes of Health (NIH) and colleagues at Science magazine to examine the issues of reproducibility and rigor of research findings. The one-day workshop brought together editors representing 30 basic- and preclinical-research journals, which had been selected by the NIH based on frequency of publication of their grantees. The objective was to discuss the measures that some journals have already taken and to agree on a common set of minimum measures to which all participants could subscribe that would be presented to the larger community for consideration. The workshop participants came to consensus on a set of principles to facilitate these goals, which has been published on the NIH website (http://www.nih.gov/about/reporting-preclinical-research.htm) along with a list of endorsing journals (http://www.nih.gov/about/endorsing-jounals.htm). NPG has, of course, endorsed these principles, as has the American Society of Gene Therapys Molecular Therapy family of journals. A key factor in data reproducibility is effective and comprehensive description of methodology. The advent of online supplements has rendered moot traditional considerations regarding the size of print publications and authors should not feel constrained by space limitations. The aim of the agreed principles is to facilitate interpretation and repetition of experiments as they were conducted in the published studies. They include guidelines for statistical analyses and for transparency in reporting key methodology, analytical information, and reagents, and mandates for data and material sharing. Editors of the MT family of journals will continue to ask reviewers to comment on whether, in their view, there is sufficient description of methodology to allow independent reproduction of the data. Many of our authors already conform to these principals, and, although these additional requirements will impose a small added burden on other authors, we are confident that, overall, the time and effort saved as a consequence of improved reproducibility will more than outweigh any inconvenience.

Escaping the Valley of Death

A joint project of the American Society of Gene and Cell Therapy (ASGCT) and the Trans–National Institutes of Health (NIH) gene therapy group, the NIH Gene Therapy Symposium took place at the NIH Natcher Center in Bethesda, Maryland, on 26–27 September 2011. More than 400 registrants, primarily from the NIH and the US Food and Drug Administration (FDA), met to review the challenges faced by investigators moving experimental gene and cell therapies into the clinic and to present examples of how technical and regulatory hurdles have been addressed both within the United States and in Europe. The genesis of the symposium was a series of meetings in February 2010 among various NIH institute directors and the then ASGCT President Ken Cornetta and Vice President Barrie Carter, with the aim of discussing how the Society and the NIH could work together to capitalize on the growing successes in gene and cell therapy. A key theme addressed at the symposium was the issue of how to cross the so-called “valley of death”—the critical period in the development pathway of complex biologics that spans the stages between preclinical validation and clinical studies of new therapeutics (Figure 1)—and how the ASGCT could work with the NIH to help facilitate clinical translation of new gene and cell therapies.

Call for papers: Nanoparticle Development and Applications in Cellular and Molecular Therapies

These are exciting times for the field of gene therapy. Various vector systems derived from naturally occurring viruses have moved to the clinic and are being used to treat human diseases as diverse as blindness, hemophilia and other hematopoietic disorders, and cancer. Despite these successes, there remains strong interest in developing nonviral vector systems with the aim of both reducing cost of therapy and mitigating toxicities, immunological side effects, and other adverse events that have occasionally been encountered with viral systems. A key mission of the Molecular Therapy family of journals—and our parent Society—is to provide a forum for presentation of emerging novel technologies for delivery of the broad spectrum of molecular therapies. It is with this in mind that we published our first Special Issue this past March, on the topic of gene editing technology and applications. Field leaders contributed cutting-edge reviews and original research on this powerful and rapidly evolving new technology. The papers are receiving robust citations and downloads, attesting to the strong interest among our readers and in the field overall.We are therefore thrilled to announce our second Special Issue, on the topic of nanoparticle development and applications in gene and cell therapy, to coincide with the 19th Annual Meeting of the ASGCT in May 2017. Nanoparticulate drug carriers and multifunctional nanoconstructs are finding increasing application in the development of cellular and molecular therapies. Nanoparticles are being engineered for controlled targeting and delivery of therapeutic agents at specific physiological sites, for in situ biological sensing (e.g., compartmental pH monitoring), and diagnostic imaging. At the same time, engineered nanoparticles can act as tools for modulating intracellular processes, facilitating study of the dynamic and integrated biochemical processes that contribute to the underlying pathogenesis of disease. Advances in material sciences have further provided myriad inorganic, organic, and composite nanosystems with exciting biophysical properties. Indeed, we have learned a great deal about how nanoparticle parameters such as shape, size, and surface characteristics modulate their biological performance.However, there remains much to be done with regard to optimization of such systems for particular physiological and therapeutic needs, particularly with respect to the type, developmental stage, and location of the disease target and the distinct biological barriers encountered at the different stages of nanoparticle targeting and delivery. This optimization should allow eventual translation into better design and engineering of safe and efficient nanopharmaceuticals and may even find application in optimization of virus-based vectors.Optimization of these systems will require further improvement of animal models representing human diseases to overcome efficacy and safety shortcomings observed in clinical trials. Furthermore, the empirical approaches often employed in nanocarrier development must be replaced by integrated “structure–activity” mapping at the single-cell, organelle, and molecular levels for better understanding of mechanistic issues. This also requires development of new and robust methodologies for precision screening. In addition, we must develop an extensive computational network knowledge of genomic and epigenomics of interindividual variations in nanopharmaceutical performance and adverse responses before pushing the boundaries of the envisaged nanotherapies to a personalized level. Finally, the proof of concept in biological targeting is not sufficient on its own; precision characterization of nanotherapeutic components is essential and should include the biophysical state of the therapeutic cargo. After all, a successful nanopharmaceutical must be structurally simple with attributes that will enable production of an affordable, viable, and clinically acceptable formulation.It is with such intentions that this Special Issue of Molecular Therapy will delve into recent advances in fundamental and translational aspects of nanoparticles in cell and molecular therapies as well as calling for a paradigm shift in the design of high-performance nanopharmaceuticals through a pan-integrated molecular bioscience and systems approach. Accordingly, we strongly encourage submission of interdisciplinary topical research, commentaries, and opinion pieces addressing these topics. This call for papers remains open until 1 December 2016 for rapid review and consideration for publication in this exciting collection. Potential contributors are encouraged to contact Guest Editors Moien Moghimi and Ernst Wagner or other members of the Molecular Therapy editorial team for rapid consideration and feedback.

Moving Forward Toward a Cure for Parkinson's: Neuropathology of the Nigrostriatal Pathway Determines the Location of Growth Factor Delivery

Vectors based on recombinant adeno-associated virus (rAAV) have been used successfully in the clinic to treat humans with Leber congenital amaurosis,1,2,3 a single-gene disorder affecting the function of photoreceptor cells of the retina, and there are ongoing clinical studies evaluating similar vectors in several disorders of the brain, including Canavan disease,4 Batten disease,5 and Parkinsons disease (PD).6 Despite these promising results, the daunting complexity of the anatomical organization of the brain challenges our capacity to deliver gene products to the precise anatomical locations where they are needed to mediate a therapeutic effect. In addition, damage to specific brain regions and the neuroanatomical pathways that connect them—such as occurs in various degenerative diseases—can further compromise our ability to deliver vectors and their therapeutic payloads. In this issue of Molecular Therapy, Ciesielska et al.7 demonstrate that degeneration of the nigrostriatal pathway in models of PD renders unfeasible therapeutic strategies based on vector delivery to the substantia nigra and instead supports a strategy based on delivery to the striatum and subsequent anterograde transport toward the substantia nigra. rAAV vectors have been shown to mediate safe, long-term gene transfer to cells and tissues of neuroectodermal origin. In particular, “first-generation” rAAV vectors—those packaged in AAV serotype 2—are quite efficient at transducing neurons in vivo. The organization of the central nervous system (CNS), comprising nuclei (neuron cell body clusters) projecting their axons along specific neuroanatomical pathways to much broader brain regions, has raised the possibility that single directed injections into confined areas might serve as “nodes” for broader and/or targeted distribution of vector or vector-expressed proteins throughout the brain. Indeed, transduction of neurons by gene transfer vectors leads to distribution of transgene and/or its gene product throughout both the dendritic arbor and axon terminals of transduced neurons. Evidence has been provided for rAAV transport along neural pathways in both the anterograde and retrograde directions.8,9 Anterograde transport is mediated by axonal transport from a neuronal cell body down the axon to the nerve terminals where axons synapse with the next neuron in the pathway. Retrograde transport denotes a process whereby vector is taken up by distal axonal termini and trafficked backward to the cell body, in the direction opposite from nerve conduction and neurotransmitter flow. PD is a progressive degenerative disorder of the CNS resulting from the death of the nigrostriatal dopamine-containing neurons of the substantia nigra. The nigrostriatal pathway, which connects the substantia nigra with the striatum, is a key pathway involved in motor control. Because this pathway degenerates in PD, the main approach to the treatment of this disorder has been to deliver neurotrophic factors such as glial cellderived neurotrophic factor (GDNF) directly to the affected striatum. GDNF is known to support the survival of dopaminergic nigrostriatal neurons, as well as others, such as motor neurons. The intent of this strategy is twofold. Any GDNF delivered to the striatum would provide therapeutic benefit to the striatal neurons themselves but could also rescue any remaining nigrostriatal dopaminergic neurons still projecting to the striatum from the substantia nigra. This is because GDNF is secreted and will benefit diseased neurons located both close to the site of viral vector delivery in the striatum and in distant regions following retrograde axonal transport. Indeed, in the healthy brain, neurons normally make use of such retrograde transport to respond to growth factors secreted by downstream targets so as to effect neuronal cell survival and growth. Although either retrograde or anterograde transport of rAAV could be useful in devising a therapeutic strategy for PD, it is crucial to know whether transport via these pathways is feasible, because the PD brain will already have suffered much damage at the time of therapeutic intervention. Of course, neither this strategy nor any other would be likely to regrow axons of neurons that have already degenerated. However, in recent years clinical trials for PD have been put forward that propose to transduce the substantia nigra with vectors expressing growth factors. This contradicts the large body of knowledge gained over many decades indicating that such an approach is unlikely to provide a clinical benefit. To address this specific issue, Ciesielska et al. made use of both rodent and nonhuman primate models in which the nigrostriatal pathway has been chemically damaged, creating a state that mimics what would be expected in the brains of humans suffering from PD. The authors found that infusion of rAAV2-GDNF vector directly into either the striatum or the substantia nigra resulted in efficient GDNF delivery directly to both sites. However, transport of GDNF to the substantia nigra occurred only via anterograde transport after striatal delivery. Direct injection of vector to the substantia nigra was not successful at delivering either vector or GDNF protein to the striatum, even though the neuron cell bodies in the reticularis portion of the substantia nigra were still intact. The findings suggest that the retrograde pathway had degenerated to such an extent that it could not transport the therapeutic toward the striatum (Figure 1). In addition, anterograde transport achieved after direct striatal delivery offers a wider distribution of GDNF to other circuitry within the basal ganglia and pallidus that is also affected in PD and ultimately may benefit from GDNF therapy as well. Thus, with this new evidence for GDNF localization to the substantia nigra and other areas of the basal ganglia after direct striatal delivery, there is little rationale for AAV2-GDNF delivery directly to the substantia nigra in PD patients who present with a degenerated nigrostriatal pathway upon diagnosis. Figure 1 Anterograde transport (right-hand arrow) of glial cellderived neurotrophic factor is blocked by loss of nigrostriatal tracks (NS track) in animal models of Parkinsons disease (PD). The “X” indicates a PD-related lesion to the anterograde ... Aside from providing key information about the therapeutic strategy for delivering neurotrophic factors to CNS sites, and specifically for confirming the gene therapy approach needed for PD, this study provides a broader lesson. Gene delivery strategies must take into account the effects of the disease state on potential target cell populations. There are numerous cases, such as in PD, in which a gene delivery strategy that could work very effectively in an uninjured organ might face significant additional barriers due to loss of cells, alteration of tissue architecture, or lack of normal organ-system function. Altered airway surface secretions in the lungs of patients with cystic fibrosis and preexisting immune cell infiltration in the livers of patients with hepatitis C virus infection are but two other examples. In any case, asking a well-designed question is vital for the success of any series of experiments, and the complex landscape of in vivo gene therapy is no exception.

And then there were two.

Molecular Therapy (MT) was conceived in the latter years of the last century as an important project of what is now the American Society of Gene and Cell Therapy (ASGCT). Now into our second decade, it has been an exciting, if occasionally bumpy, ride. Launched during the heady early days of the field of gene therapy, there were those who might have thought it risky to start a brand new journal during a period of uncertainty for the business of science publishing. However, thanks in no small part to the vision and efforts of the journal’s early Editors-in-Chief (EiCs) and the tremendous support of the Society and its members, we find ourselves thriving more than 10 years on. Institutional subscriptions to the journal have never been higher, and submissions continue to climb. And in 2010 our impact factor hit 7.15, allowing us to compete ever more effectively with a host of high-quality journals serving the various subfields falling under the molecular therapy umbrella. It is in this context that we are thrilled to announce the launch of a new sibling journal, Molecular Therapy—Nucleic Acids (MTNA), which will operate under a Web-only, fully open-access model. We are equally thrilled to announce that John Rossi will serve as the inaugural EiC of the new journal. John has served as an adviser to MT since its inception and currently serves as a Deputy Editor; his involvement and leadership will greatly facilitate the development of MTNA.