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Comparing Off-Target Editing Rate with the Natural Mutation Rate of the Human Genome It is important to note that accurate assessment of the specificity of a genome-editing approach requires that mutations created by the genome-editing process be distinguished from those that occur spontaneously throughout a life span treatment breast cancer order cheap prometrium online. A direct comparison between the mutation frequency generated by a genome editing nuclease and the spontaneous mutation frequency has not yet been conducted treatment pneumonia buy discount prometrium 200 mg on-line, but results from this type of analysis are likely to depend on the specific nuclease in question and on which cell type is examined medicine articles purchase prometrium pills in toronto. The error rate of nuclease technologies continues to improve and may at some point treatments generic prometrium 100mg, if it is not already medicine 751 m discount 100mg prometrium amex, be less than the spontaneous mutation frequency symptoms 11 dpo prometrium 200 mg on-line. Measuring Efficiency and Specificity for Each Delivery Platform For genome-editing applications, the system (nuclease and targeting sequences) must be delivered inside cells. Because the choice of delivery platform determines the extent, level, and time course of expression of the genome-editing machinery, it affects the efficiency and specificity displayed in a given set of experimental conditions and furthermore determines the toxicity and immunogenicity profile. Finally, the frequency with which the intended target sequence and related sequences occur in the genome and the local chromatin environment at the target site also can influence the efficiency and specificity of a genome-editing approach. All the factors mentioned above are likely to vary according to the treated cell type and modality (ex vivo versus in vivo). The design of these preclinical studies is influenced by the choice of target cells and experimental conditions, and the results should be viewed as providing relative rather than absolute values. An additional caveat is that most of these preclinical studies measure nuclease activity and specificity over a large population of cells, among which nuclease expression will vary. Because the ratio of on to off target activity also varies with nuclease expression level, the cells with higher nuclease expression may have a less favorable ratio since the on-target activity will saturate, while activity at off-target sites becomes more evident. On the other hand, cells with lower expression may exhibit a more favorable ratio because activity is evident mainly at the intended target site. This consideration suggests that the dose dependence of on and off-target rates be considered as part of the process of validating a genome-editing approach. Assessment of nuclease specificity will continue to evolve as scientific knowledge and techniques improve. From an operational standpoint as of this writing (late 2016), however, the following represents a reasonable approach to conducting this assessment: Use both bioinformatics and unbiased screens to identify potential off-target sites (see Appendix A). It should be noted that most off-target sites identified to date lie in non-protein coding regions of the genome, making their functional importance difficult to assess. It is important to note that to develop a genome-editing approach for clinical use, it may not be necessary or feasible to conduct comprehensive efficiency and specificity studies performed at high-enough sensitivity to capture all possible off-target edits. Ongoing work in standard gene therapy, for example, has indicated that uncontrolled lentiviral insertions, which cause even more disruptive changes than non-homologous repair of a double-strand break, may be relatively safe and well tolerated in several types of cells and tissues. This is true even when large numbers of 8 9 insertions (up to 10 or 10 per patient) are introduced. A further consideration is that the off target activity is dependent on the sequence. Much of the early preclinical testing aimed at establishing targeting efficacy and specificity has been carried out in nonhuman organisms, especially mice. However, the genomes of humans and mice are sufficiently divergent that assessment of the specificity of engineered nucleases in the genomes of mice or other rodents may have somewhat limited predictive power for the same genome-editing approach in humans. In the end, therefore, each strategy will need to be evaluated against the others in terms of efficacy, risk, cost, and feasibility. These regulatory systems include a wide range of preclinical models and study designs to support the clinical development of therapies based on edited cells, as well as a roadmap for first-in-human clinical testing and eventual marketing. Other countries have similar pathways, as described in Chapter 2, albeit with some variations in the stage of research at which a cell-based therapeutic can be marketed and the terms under which it can be withdrawn. The question of approval for clinical use hinges largely on identifying when benefits may be expected to outweigh risks when used as labeled and as intended (Califf, 2017). Clinical trial data is increasingly reviewed within a structured framework that identifies need, alternatives, 44 areas of uncertainty, and avenues for risk management. They must consider the existence and effectiveness of alternative treatments, disease severity, risk tolerance of affected patients, and potential for additional insight from postmarket data. Long-term follow-up is not always required, for example when preclinical data on such things as vector sequence, integration and potential for latency demonstrates that long-term risks are very low. But when long-term risks are present, a gene therapy clinical trial must provide for long-term follow-up observations in order to mitigate those risks. Where merited, the guidance suggests a 15-year period of post-trial contact, observation and physical exams (though this can be shortened based on factors such as vector persistence, or when subjects are predicted to have only short-term survival). Prior to enrolling, subjects must give voluntary, informed consent to long-term follow-up, and while they may withdraw at any time, it is hoped they will comply. Off-label use of cells subjected to genome editing would be legal in the United States, in Europe, and in other countries, and is probably to be expected with respect to patient populations (for example, if approved for adults, use might well be extended off-label to pediatric 45 populations) or for varying degrees of severity of the disease indication. The prospect of off label use has led to speculation about uncontrolled expansion of the technology into uses that are unsafe, unwise, unnecessary, or unfair. And it is true that off-label use, while an important aspect of innovative medicine, can at times lead to uses that lack a rigorous evidentiary basis. But the specificity of these edited cells may limit the range of off-label uses for unrelated indications 46 more than is the case with many drugs. While one might imagine a cell therapy based on genome editing for muscular dystrophy being of possible interest to those with healthy muscle tissue who wish to become even stronger, other examples are more difficult to envision, at least for the near future. This point is of particular relevance to concerns about uses that go beyond restoration or maintenance of ordinary health (discussed in Chapter 6) because the specificity of edited cells will make such applications less likely at this time. Several technical challenges faced in moving somatic genome editing toward clinical testing have already been met by conventional somatic gene therapy. Concerning ex vivo strategies, they are based on modifying human cell types and thus can be tested only in in vitro culture models or upon xenotransplant of the modified cells into immunocompromised mice. These studies interrogate cell viability, biodistribution, and biological function in vivo, including self renewal, multipotency, and clonogenicity, all crucial features of stem cells. In vivo strategies may require preclinical testing of toxicity and biodistribution in nonhuman primates, including evidence that unintentional modification of the germline does not occur. Note, however, that most assays of germline transmission have low sensitivity, and thus a certain degree of uncertainty may have to be managed in considering clinical development and regulation. Several guidance documents have been published by regulatory authorities in the U. Such guidelines may be suitably adapted to design preclinical studies of somatic genome editing strategies. The International Standards Coordinating Body was established to advance process, measurement, and analytical techniques to support the global availability of cell, gene, tissue-engineered, and regenerative medicine products, and cell-based drug discovery products. Creating standards creates a more uniform compliance environment and addresses and assists in future efforts for harmonization internationally of the regulatory framework for 47 submissions across the globe. Regulating Somatic Genome Editing by Approach and Indication An ethical and regulatory assessment of future somatic genome-editing applications may depend on both the technical approach to the editing and the intended indication. Like traditional gene therapy, somatic genome editing could be used to revert an underlying genetic mutation to a variant not associated with disease, which would result in a fraction of the targeted cells regaining normal function. Somatic genome editing also could be used to engineer a cell so that its phenotype differed from that of a normal cell and was better able to resist or prevent disease. For example, a cell could be changed so that it made above-normal amounts of a protein, or so that it was resistant to a viral infection. Both ex vivo and in vivo approaches to genome editing could be applied to treat or prevent a disease. In addition, genome editing could be used to alter a trait not associated with disease (see Chapter 6). Regardless of the final framework used to assess human somatic cell genome-editing applications, it is vital that the regulatory oversight mechanisms have sufficient legal authority and enforcement capability to identify and block unauthorized applications. To date, the existing structures have been successful in preventing unauthorized applications of gene therapy and the current framework provides guidance on key elements. Although human genome editing may be somewhat more difficult to control than traditional gene therapy because technical advances have made the editing steps easier to perform, the cellular manipulations and delivery of edited cells to the patient continue to demand high-quality laboratory and medical facilities, which generally will ensure that regulatory oversight is in place. Overall, then, regulatory bodies need the legal authority, leadership commitment, and political support to apply their legal powers to halt the marketing of therapies that use human genome editing products that have not undergone regulatory review and approval (Charo, 2016b). Special Considerations Associated with Genome Editing in Fetuses In certain situations, either the most effective or the only approach would be to attempt to edit the somatic cells of a fetus prior to delivery. Diseases for which these special circumstances might apply include those that are multisystemic or have an extremely early onset that would make postnatal intervention too late to benefit the child or are extremely challenging from a technical standpoint. In addition, because of the tremendous developmental plasticity of the fetus, fetal editing might be more effective than postnatal editing in certain circumstances. An example would be attempting to revert a disease-causing variant that affects every neuron in the brain. In a more general sense, the therapeutic editing process could be carried out ex vivo in a scenario in which cells could be harvested from the fetus, edited outside the body, and then transplanted back into the fetus. Currently, established methods for isolating and transplanting autologous fetal cells are available for a limited number of cell types, but the range of cell types is likely to increase in the future. Therapeutic editing in fetuses also could be performed in vivo, in which case the editing machinery would be delivered to the fetus to modify cells in situ. As noted above, the in situ correction of a disease-causing variant early in development has the potential to be more effective than postnatal in vivo editing, when many organ systems are more fully developed. In utero stem cell therapy has been tried (with limited success) (Couzin-Frankel, 2016; Waddington et al. Although fetal genome editing has potential advantages, at least two special ethical issues would need to be addressed: special rules for consent (see Chapter 2) and the increased risk of causing heritable changes to the germline by causing modification of germ cells or germ cell progenitor/stem cells. With regard to consent, key issues have been addressed by existing oversight mechanisms, fetal surgery has already been used in clinical care, and in utero fetal gene therapy is attracting increasing interest (McClain, 2016; Waddington, 2005). The risk/benefit calculation is shifted relative to a postnatal or adult intervention, with the degree of risk to which a fetus can be subjected being strictly limited when there is no prospect of medical benefit to the future child. When such benefit is possible, however, the more usual standards for risk/benefit balance apply. Decisions about fetal surgery have been made with the understanding that the pregnant woman has the ethical and legal authority to give informed consent. In the United States, as in other countries, maternal consent is required (Alghrani and Brazier, 2011; OConnor, 2012), and when 50 research is aimed at maternal health as well, maternal consent alone is sufficient. A second issue is the challenge of assessing whether unintended germline editing has occurred if in vivo somatic editing is attempted in a fetus. A key feature of germline cell development is that the primordial cells that will give rise to germ cells are sequestered from somatic cells at key developmental points. Before this sequestration of germline and somatic cells occurs or has been finalized in early development, germline cells might be edited as efficiently as would be the desired somatic cell targets. As a result, there could be a higher risk of unintentional edits to germline cells early in fetal development compared with performing the same intervention later in fetal development. It might be possible only to assess postnatally whether editing of germ cells or germ cell progenitors had occurred, at which time it would be too late to change the outcome. Human genome editing in somatic cells holds great promise for treating or preventing many diseases and for improving the safety, effectiveness, and efficiency of existing gene therapy techniques now in use or in clinical trials. The ethical norms and regulatory regimes already developed for gene therapy can be applied for these applications. Regulatory assessments associated with clinical trials of somatic cell 49 See. Regulatory oversight also will need to include legal authority and enforcement capacity to prevent unauthorized or premature applications of genome editing, and regulatory authorities will need to continually update their knowledge of specific technical aspects of the technologies being applied. Existing regulatory infrastructure and processes for reviewing and evaluating somatic gene therapy to treat or prevent disease and disability should be used to evaluate somatic gene therapy that uses genome editing. Oversight authorities should evaluate the safety and efficacy of proposed human somatic cell genome-editing applications in the context of the risks and benefits of intended use, recognizing that off target events may vary with the platform technology, cell type, target genomic location, and other factors. Transparent and inclusive public policy debates should precede any consideration of whether to authorize clinical trials of somatic cell genome editing for indications that go beyond treatment or prevention of disease or disability. Thousands of genetically inherited diseases are caused by 52 mutations in single genes. While individually, many of these genetically inherited diseases are rare, collectively they affect a sizable fraction of the population (about 5-7 percent). The emotional, financial, and other burdens on individual families that result from transmission of such serious genetic disease can be considerable, and for some families could potentially be alleviated by germline editing. Recent advances in the development of genome-editing techniques have made it realistic to contemplate the eventual feasibility of applying these techniques to the human germline. As discussed elsewhere in this report, improvements in genome-editing techniques are driving increases in the efficiency and accuracy of genome editing while also decreasing the risk of off-target events. Because germline genome edits would be heritable, however, their effects could be multigenerational. As a result, both the potential benefits and the potential harms could be multiplied. In addition, the notion of intentional germline genetic alteration has occasioned significant debate about the wisdom and appropriateness of this form of human intervention, and speculation about possible cultural effects of the technology. As discussed below, these include concerns about diminishing the dignity of humans and respect for their variety; failing to appreciate the importance of the natural world; and a lack of humility about our wisdom and powers of control when altering that world or the people within it (Skerrett, 2015). The distinction turns on intent rather than on the technological intervention, which is highly similar in both cases. Sons could not pass along the donated mitochondria to their own future children, but daughters could, through their now-modified eggs, thus rendering this a potentially heritable form of germline alteration. This chapter begins by reviewing potential applications of and alternatives to heritable genome editing. It then describes in turn scientific and technical issues, ethical and social issues, and potential risks associated with these applications.

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The exam may require recognition of common as well as rare clinical problems for which patients may consult a certified internist. Trainees, training program directors, and certified practitioners in the discipline are surveyed periodically to provide feedback and inform the blueprinting process. Questions ask about the work done (that is, tasks performed) by physicians in the course of practice: Making a diagnosis Ordering and interpreting results of tests Recommending treatment or other patient care Assessing risk, determining prognosis, and applying principles from epidemiologic studies Understanding the underlying pathophysiology of disease and basic science knowledge applicable to patient care Clinical information presented may include patient photographs, radiographs, electrocardiograms, recordings of heart or lung sounds, and other media to illustrate relevant patient findings. Below each major category are subsection topics and their assigned percentages in the exam. Its cause remains unknown, despite evidence that genetic, environ mental, and immunological factors may play a role in its pathogenesis. Brain tissues from cer ebellum, midfrontal, and cingulate gyrus obtained at autopsy from 11 patients with autism were used for morphological studies. We demonstrate an active neuroinflammatory process in the cerebral cortex, white matter, and notably in cerebellum of autistic patients. Our findings indicate that innate neuroimmune reactions play a pathogenic role in an undefined proportion of autistic patients, suggesting that future therapies might involve modifying neuroglial responses in the brain. Neuropathological studies have in 30% of affected children, usually between 18 and 24 shown that abnormalities in cytoarchitectural organiza 1 months. They suggest that syndromes, although in most patients the causes are defects in neuronal maturation and cortical organiza 3,4 still unknown. The potential role for maternal antibodies and determined the magnitude of neuroglial and in 19 as a pathogenic factor also has been proposed. Despite the growing interest in possible Patient Information immune mechanisms in its pathogenesis, there has Brain tissues from autistic patients and nonneurological con been no direct evidence linking findings in the periph trol cases were obtained through the Autism Tissue Program eral blood to immune activity in the brain of autistic of the Harvard, University of Miami, and University of 21 Maryland Brain Banks. Only a few reports have described gliosis and 12,22 a criterion for inclusion in the repository. The primary antibodies and dilutions are described in brains from autistic patients had fresh-frozen tissues available Table 3. Fresh-frozen tissues from four other cases of autism, and six control cases in which only frozen tissue was available were included for protein analysis. This method measures the percentage of the area years) were collected by lumbar puncture during conscious of interest that is immunoreactive for a specific antibody. Confocal Microscopy were quantified in tissue homogenates by sandwich enzyme Formalin-fixed brain tissues were cryoprotected with sucrose immunoassay using commercially available kits according to solutions and then cut with a sliding microtome to yield the manufacturers protocols. Proteins Included in the Cytokine Protein priate fluorogen-tagged secondary antibody (Cy3 or Alexa). Because of the subset of autistic (n 7) and control patients (n 7) nonparametric nature of the data (as determined by tests of from whom fresh-frozen brain tissue had been obtained normality), nonparametric tests were used to increase the ro (see Table 1). The most prominent microglial reaction was ob were used because they make no assumptions about the dis served in the cerebellum, where the immunoreactivity tribution of the data (eg, normality). Further immuno responses characterized by microglial and astroglial ac cytochemical studies, including confocal microscopy, tivation. We observed no differ showed increased astroglial reactions characterized by ences in microglial or astroglial activation as a function an increase in the volume of perikarya and glial pro of age or clinical profile including history of develop cesses. The magnitude of astroglia reaction measured by some cases panlaminar astrogliosis was observed (Fig area fraction of immunoreactivity or Western blot was 2). Quantitative assessment of astroglial immunoreac similar in autistic brain tissues from patients with and 25,26 tivity by fractional area methods showed a signifi without history of epilepsy. Lack of Evidence of Adaptive Immune Reactions in dence of leptomeningeal, parenchymal, or perivascular Autistic Brains inflammatory infiltration in autistic brains in any of the To examine more closely the immunopathological reac regions studied. Immunostaining with antibodies recog tions associated with adaptive immunity in the brain of nizing IgG, IgA, or IgM showed no deposition of any of autistic patients, we performed immunocytochemical these immunoglobulins in neuronal or neuroglial cell studies to identify T and B-lymphocyte infiltration and populations. In cerebella from autistic brains, we ob deposition of immunoglobulin and complement, as in served deposition of complement membrane attack com dicators of cellular and humoral immune responses. Similar clusters of microglia (F) and astrocytes (H) visualized with diaminobenzidine tetrahydrochloride chromogen. Interestingly, a larger spectrum tients from whom fresh-frozen brain tissues were avail of increased proinflammatory and modulatory cyto able (see Table 1). Each spot represent a cytokine for which the ratio of expression (arbitrary units) was obtained between the cytokine and the positive control present in the membrane. Innate Immune Responses in Autism In this study, we have demonstrated a marked increase the Cerebrospinal Fluid from Patients with Autism in neuroglial responses, characterized by activation of Shows a Proinflammatory Profile microglia and astroglia, in the brains of autistic pa Because brain tissues from patients with autism showed tients. In ders (eg, pseudotumor cerebri or headaches; see Table our sample of autistic cases, microglial and astroglial 2). Quantitative validation analysis by enzyme-linked immunosorbent assay and immunolocalization of cytokine expression in the brain. The neuroglial activation in the autism brain tissues was particularly striking in the cerebellum, and the changes were associated with upregulation of selective cytokines in this and other regions of the brain. Im munocytochemical analysis of microglial and astroglial reactions in the brains of these patients showed that regardless of age, history of epilepsy, developmental re gression, or mental retardation, marked morphological changes consistent with chronic and sustained neuro glial inflammatory responses were present in cortical and subcortical white matter as well as in the cerebel lum. These changes may be involved in mechanisms associated with neuronal and synaptic dysfunction in autism. Vargas et al: Neuroglial Activation in Autism 77 may be involved in the mechanisms of neuronal and mary or secondary responses may be valuable clinical synaptic dysfunction. These observations suggest that the adaptive both proinflammatory and antiinflammatory cytokines immune system does not play a significant pathogenic 45 as well as growth and differentiation factors. Because our of the brain: as a direct effector of injury and on the study focused on autopsy tissues, we cannot exclude 46 other hand as neuroprotectant. An issue that remains the possibility that specific immune reactions, mediated unclear is how and when microglia and astroglia be by T-cell and/or antibody responses, occurred at the come activated in the brain of autistic patients. Neu onset of disease, during prenatal or postnatal stages of roglial responses in autism may be part of both primary development. The lack of immu vated microglia in the brain in autism may reflect ab noglobulin deposition, however, suggests that comple normal persistence of fetal patterns of development in ment activation may occur in the absence of antibody response to genetic or environmental (eg, intrauterine, mediated pathways and may resemble the maternal) factors. Further clarification of the role of series of cases are needed to clarify these issues. An alternative explanation is that showed the most prominent neuroglial responses. In may suggest an association with maturation of Purkinje 60 stead, our observations suggest that the pathological cells. Furthermore, this process continues beyond early cortex and cerebellum of autistic brains may have im neurodevelopment and is present even at very late stages portant implications for the neurobiology of autism. It can suppress Purkinje cells plays a role in the etiopathogenesis of au specific immune responses by inhibiting T-cell prolif 53 tism. These findings suggest that the elevation of this elevated in the brain regions studied. It could be that cytokines derive from neuroglial and neuronal sources as demonstrated by our immunocyto References chemical assessment. Heritable and nonheritable velopmental arrest because some of the cytokines are risk factors for autism spectrum disorders. Genetics of autism: complex ae glial reactions, in the form of innate immune re tiology for a heterogeneous disorder. Genetic and im bellum is the focus of an active and chronic neuroin munologic considerations in autism. Circulating autoantibod flammation in the context of the genetic and other fac ies to neuronal and glial filament proteins in autism. New insights into neuron-glia cytokine production associated with innate and adaptive im communication. J Neuroimmunol 2001;120: astrocytes: redefining the functional architecture of the brain. Cytokines and acute neurodegenera tibodies associated with autism and a language disorder. Innate immune recognition and creased in the cerebrospinal fluid of patients with ischemic control of adaptive immune responses. Microglia in de tribute to non-lymphocyte-mediated brain disease induced by generative neurological disease. Cytotoxicity of mi autism: the role of the dorsal medial-frontal cortex and anterior croglia. Glial cells and neurotransmission: an acute and stable disease and undergoing immunomodulatory inclusive view of synaptic function. Legal Responsibilities of Designated Aviation Medical Examiners Title 49, United States Code (U. Approximately 450,000 applications for airman medical certification are received and processed each year. It is essential that Examiners recognize the responsibility associated with their appointment. In this situation, both the applicant and the Examiner in completing the application and medical report form may be found to have committed a violation of Federal criminal law which provides that: "Whoever in any matter within the jurisdiction of any department or agency of the United States knowingly and willfully falsifies, conceals, or covers up by any trick, scheme, or device a material fact, or who makes any false, fictitious or fraudulent statements or representations, or entry, may be fined up to $250,000 or 6 Guide for Aviation Medical Examiners imprisoned not more than 5 years, or both" (Title 18 U. In view of the pressures sometimes placed on Examiners by their regular patients to ignore a disqualifying physical defect that the physician knows to exist, it is important that all Examiners be aware of possible consequences of such conduct. Reports regarding the medical status of an airman should be written by their treating provider.

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