Appendices
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Appendix 1. The Transfer of Human Stem Cells or their Direct Derivatives into Animal Hosts
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Recommendation A6.1: Research involving the transfer of human stem cells or their direct neural and/or glial derivatives into the central nervous systems of postnatal animal hosts requires review by institutional animal research oversight committees supplemented by reviewer expertise in stem cell or developmental biology.
For protocols involving the transfer of human stem cells or their direct neural and/or glial derivatives such that they contribute to the central nervous systems of postnatal animal hosts, research review should be conducted by animal research review committees supplemented with expertise in stem cell or developmental biology. Review should be based on a reasonable extension of existing animal research standards, which are themselves based on rational, practical, fact-based assessments of the effects of research manipulations on cognitive and functional outcome measures, as well as on animal health and welfare.
Additional data collection and monitoring by animal research committees should be commensurate with the anticipated characteristics of the modified animal in the context of the proposed research. Issues regarding the possible change in or enhancement of an animal’s behavior or operationally-assessed cognition should be addressed through diligent application of accepted principles for the humane treatment and protection of animals in research, and primarily through regular animal research oversight mechanisms.
Monitoring and data collection should be based upon a sound assessment of the developmental trajectories of the animal host that may be further affected by taking into account the environmental and epigenetic context in which the donor genes or cells are going to be deployed. It should be grounded in existing knowledge of such trajectories, with reasonable scientific inferences as to their phenotypic and fate potential, with thorough reference to the physiological and behavioral tests and assessments currently available by which to assess the host species.
Research involving the modification of the central nervous system, as established with the introduction of human stem cells or their neural and/or glial derivatives in a way that they contribute to the brains or spinal cords of animal hosts, may attempt to model or directly mimic aspects of human neurological and neuropsychiatric function. As such, this research may demand specialized cognitive and behavioral assessments of the sort conducted in neuroscientific research. There may be an irreducible degree of uncertainty about the internal cognitive processes of any new animal model, in particular how it would manifest distress, anxiety, or other aspects of animal welfare. In such cases, as with transgenic animals, researchers and institutions should familiarize themselves with available options for behavioral response assessment. A baseline of normal behavioral data for the test species and strain should be available before experimentation is permitted, so as to enable clear and rapid identification of behavioral differences or abnormalities associated with treatment and/or human cell transfer. Investigators and institutions should also consider requiring limited pilot studies to produce initial data on the effects of experimental interventions on modified animals, monitoring all deviations from normal behaviors, with prescribed discussion with pertinent animal welfare committees before proceeding to definitive experiments.
Investigators and institutions should also make appropriate adjustments to research protocols to take into account new data or unanticipated responses from animal subjects that may inform or alter the continued permissibility of the animal’s participation in the study. These include identifying any novel signals suggesting a material change in an animal’s condition, comfort, or behavioral state or repertoire, whether by way of deterioration or enhancement. Regular reassessment of animal welfare during the course of experimentation is essential.
Research with a known, intended, or well-grounded significant potential to create some aspect suggestive of human cognition, self-awareness, behavior or behavioral pathology, while not prohibited, should be subject to close scrutiny, taking care to ensure the humane protection of animal subjects. Such studies require a clear and compelling justification, grounded in the potential for significant scientific breakthrough, clinical advance, or both.
Through retained advisors or committee diversity, animal research review committees should ensure that they have sufficient scientific and clinical expertise to make appropriate judgments concerning the matters discussed in these recommendations.
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Recommendation A6.2: Researchers using large, complex animal models, such as livestock and non-human primates, should follow international standards for livestock animal and non-human primate research, which call for frequent monitoring of animals whenever there is the potential for unexpected outcomes and unanticipated phenotypes.
Best practices dictate that research with non-human primates (NHPs) should account for the following (Tardif et al. 2013):
Investigators must justify their choice of an NHP species in light of the goals of the study.
For some NHP species, temporary removal of an individual from its social group may cause it acute stress, and permanent removal may cause distress (the inability to cope with stress). Because of this variability, investigators and veterinarian staff must be aware of normal behaviors of individual NHPs and must know how to identify potential signs of stress and distress.
Because NHPs are valuable experimental subjects, they are often used in serial studies. Both the number of procedures and their consequent levels of burden must be strongly justified by investigators and monitored by trained veterinary staff.
Housing NHPs in social groups best replicates the social interactions they experience in the wild and thereby promotes species-typical behaviors and psychological well-being. For this reason, any singly housed modified NHPs should kept so for the minimum length of time required. The need for single housing should be reviewed by animal research committee members and veterinary staff. Because NHPs are social animals, single housing can produce a reduced range of species-typical behavior, increased environmental stressors, and self-inflicted wounding or withdrawn behavior. Not only could these outcomes affect the welfare of modified NHPs, but they might also confound investigators’ judgements about any potential behavioral changes caused by the transfer per se of human stem cells or their direct derivatives.
Best practices dictate that research with livestock animals should abide by the following standards:
Guide for the Care and Use of Laboratory Animals – guidance document required for PHS funded studies in US and for AAALAC accredited facilities worldwide https://www.aaalac.org/the-guide/
European standards, also a core reference resource for AAALAC https://www.aaalac.org/pub/?id=E900C0A9-EEB3-1C2E-6A3C-0C84FF348CDD
Research with Agricultural Animals and Wildlife. ILAR Journal, Volume 60, Issue 1, 2019, Pages 66–73. https://academic.oup.com/ilarjournal/article/60/1/66/5490285
Agricultural Animals as Biomedical Models: Occupational Health and Safety Considerations. ILAR Journal, Volume 59, Issue 2, 2018, Pages 161–167 https://academic.oup.com/ilarjournal/article/59/2/161/5196514#140647793
Appendix 2. Sample Informed Consent Documents for Procurement of Human Biomaterials for Stem Cell Research
Appendix 3. Informed Consent Considerations for Procurement of Cells and Tissues for Stem Cell Research and Translation
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The informed consent process for the procurement of cells and tissues for stem cell research and translation should cover the following statements, adapted to the particular project:
That the cells and tissues may be used in the derivation of continuously growing cell cultures, including production of embryonic or pluripotent stem cell lines.
That the embryos or tissues will be destroyed, or isolated cells altered, during the process of deriving totipotent or pluripotent cells for research.
That derived cells and/or cell lines might be deposited and stored in a repository many years and used internationally for future studies, many of which may not be anticipated at this time.
That cells and/or cell lines might be used in research involving genetic alteration of the cells, the generation of organoids (small organ models) or animal research (resulting from the transfer of human stem cells or their direct derivatives into animal models, or the introduction of human stem cells into animal embryos).
That the donation is made without any restriction or direction regarding who may be the recipient of transplants of the cells derived, except in the case of autologous transplantation or directed altruistic donation.
Whether the donation is limited to specific research purposes or is for broadly stated purposes, including research and/or clinical application not presently anticipated, in which case the consent shall notify donors, if applicable under governing law, of the possibility that permission for broader uses may later be granted and consent waived under appropriate circumstances by a human subjects review committee. The consent process should explore and document whether donors have objections to the specific forms of research and/or clinical application outlined in the research protocol.
Whether the donor may be approached in the future to seek additional consent for new uses or to request additional materials (such as blood or other clinical samples) or information.
Disclosure of what donor medical or other information and what donor identifiers will be retained, specific steps taken to protect donor privacy and the confidentiality of retained information, and whether the identity of the donor will be readily ascertainable to those who derive or work with the resulting stem cell lines, or any other entity or person, including specifically any oversight bodies and government agencies.
Disclosure of the possibility that any resulting cells or cell lines may have commercial potential, and whether the donor will or will not receive financial benefits from any future commercial development.
Disclosure of any present or potential future financial benefits to the investigator and the institution related to or arising from proposed research.
That the research is not intended to provide direct medical benefit to anyone including the donor, except in the sense that research advances may benefit the community.
That neither consenting nor refusing to donate biomaterials for research will affect the quality of care provided to potential donors.
That there are alternatives to donating human biomaterials for research, and an explanation of what these alternatives are.
For donation or creation of embryos for research, that the embryos will not be used to attempt to produce a pregnancy.
For donation of gametes, that they will not be used to create embryos unless explicit consent is obtained and the resulting embryos will not be used for reproductive purposes.
For experiments in embryonic stem cell derivation, somatic cell nuclear transfer, somatic cell reprogramming, parthenogenesis, or androgenesis, that the resulting cells or stem cell lines derived would carry some or all of the DNA of the donor and therefore be partially or completely genetically matched to the donor.
That nucleic acid sequencing of the resulting stem cell line is likely to be performed and this data may be stored in databases available to the public or to qualified researchers with confidentiality provisions, and that this may compromise the capacity for donation to remain anonymous and/or de-identified.
That the donor and/or biomaterials will be screened for infectious and possibly genetic diseases or markers of disease.
Whether there is a plan to share with the biomaterials donor any clinically relevant health information discovered incidentally during the course of research, and if so, what those plans are, including the right not to receive such results.
Appendix 4. Sample Material Transfer Agreement Document
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Download the ISSCR SAMPLE FORM Microsoft Word document: Uniform Human Embryonic Stem Cell-related Materials Transfer Agreement
Appendix 5. Considerations for Genome Editing Research
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Assessing tumorigenicity of genome-modified cell interventions
For gene-modified cell products early readouts of a potential tumorigenic risk could include the expansion of one or few dominant clones from a starting polyclonal graft in a (xeno)-transplanted host. The emergence of such dominant clones may highlight the occurrence within the administered cell population of some genotoxic events consequent to the genetic modification, such as integration of a gene transfer vector or editing-induced translocation nearby an oncogene. These random and presumably rare events may activate the tumorigenic potential of the oncogene and endow the affected cell with a gain-of-function mutation promoting its growth. It should be realized that cells carrying genotoxic events leading to a gain of function may not progress to the formation of a full-blown tumor in preclinical models for lack of proper supporting conditions, sufficient follow-up time or the small scale of the study. On the other hand, such could develop in humans, where more cells are administered and clinical persistence may extend far longer than in conventional preclinical models. Clonal tracking of administered cells in vivo has been primarily developed and validated as a safety readout in the field of hematopoietic stem cell gene therapy, where semirandom genome-wide insertions of vector provide a unique clonal marker of transduced cells. In studies using early generation retroviral vectors, expansion of rare clones carrying vector integration nearby certain oncogenes was often reported, both in animal models as well as in human subjects, and some of these clones eventually progressed to overt leukemia. In these cases, identification of a vector insertion next to an oncogene in the leukemic clone allows tracing the origin of the leukemia to the original genetic modification. Such clonal markers of gene-modified cells may not be available when using other engineering platforms such as genome editing or when the cell product does not undergo genetic engineering. Clonal tracking could still be attempted using surrogate readouts such as randomly acquired genomic mutations and monitoring the graft for the skewing from polyclonal to oligoclonal composition and the potential emergence and expansion of clones having a growth advantage, which may eventually progress to tumors.
Preclinical Safety and Efficacy Involving Genome Modified Cell Interventions
The following must be addressed and minimized through preclinical studies before initiating a first-in-human clinical trial.
Issues Particular to Gene Replacement
Semi-random insertion of exogenous DNA may cause genotoxicity when a sporadic insertion takes place near an oncogene and causes its activation by truncation and/or transcriptional activation or disrupts tumor suppressor genes. These events may be rare but due to the very large number of insertions typically occurring in some cell therapies, they may well occur within a cell product. The few cells bearing such insertions might then expand and become dominant in vivo because of the enhanced growth potential afforded by the mutation. Genome insertions are expected with integrating vector platforms (such as retro-/lenti-viral vectors or transposons) but may also occur inadvertently and to lower extent when episomal DNA (i.e. from AAV vectors or plasmids) become incorporated by non-homologous end joining (NHEJ) at sites of DNA double-stranded breaks (DSB). For integrating vectors, a design should be used that minimizes the risk of genotoxic insertions (i.e. reducing the extent of transcriptional transactivation or readthrough from insertion site). Some knowledge should also be acquired on the genome-wide insertion pattern in the selected cell type and any existing specific biases that may increase the risk of genotoxic insertions. The genomic distribution of vector insertion should be assessed by preclinical studies in the cultured treated cells as well after in vivo administration into recipients, which should be monitored for the emergence of dominant clones with genotoxic insertions. Information available from prior studies performed with the same or similar vector backbone and target cells might alleviate the requirement for new extensive investigation. For non-integrating platforms, the residual extent of insertion or lack thereof should be investigated or previously known.
The potential mobilization of the vector, whether integrated or maintained as an episome, upon superinfection of the engineered cells in the recipient by wild-type virus, and the possibility of recombination of the vector genome with the wild-type viral genome should also be considered among the potential long-term risk. It is expected that recombination of vector sequence with the parental viral genome would most often result in a replication-defective virus. However, the potential risk of incorporating a new and biohazardous gene in the viral gene pool should be considered and, if present, alleviated by adopting conditions minimizing such risk. Many integrating vectors derived from retro-/lenti-viruses are commonly designed to be “self-inactivating”. This design means that upon integration the viral long terminal repeats are deleted of most transcription activating sequences. Such deletion makes the rescue of proviral expression, and its capture by the superinfecting virus, highly unlikely.
Cytoplasmic and nuclear exposure to exogenous nucleic acids, whether of viral, plasmid or other origin, and their replication intermediates might activate the innate immune sensing machinery in the treated cells. This activation may in turn trigger detrimental and inflammatory responses, potentially spreading to neighboring cells. Such responses might be minimal and only have subtle effects. However, if their activation is more robust or sustained they might impact the ability to engraft and adversely affect the clonal composition and long-term stability of an engineered cell graft. Importantly, these responses might be substantially augmented by excess impurities, such as DNA fragments and residual plasmid in the final cell product. Thus, efforts should be made to reduce impurities in the vector preparation.
Pre-existing immunity to viruses used to make gene transfer vectors may limit their application in vivo. This might be due to the presence of high-titer neutralizing antibodies in the plasma that may inactivate the vector and thus block gene transfer. Another possibility is the recognition of residual viral components in the transduced cells by T-cells, which may lead to the immune-mediated clearance of the transduced cells. The latter response might also affect ex vivo engineered cells if administered shortly after vector exposure. These immune responses may impact the in vivo survival of engineered cells and should be appropriately investigated before clinical testing.
Issues Particular to Genome Editing
The first and best developed approach to genome editing exploits engineered endonucleases to deliver a DNA double-stranded break (DSB) to the intended target sequence. One main safety concern is the off-target activity of the nuclease. Extensive preclinical testing should be performed to interrogate the genome-wide specificity of the editing reagents using orthogonal techniques. The target sequence is first chosen to be uniquely represented in the genome and with limited or no occurrence of any highly similar homologous sequences bearing only a few mismatches. Bioinformatic prediction of potential off targets is then performed to rule out potential activity in known sensitive genomic sites (such as tumor suppressor genes). An experimental assessment of specificity is then performed on DNA in vitro or in cell lines exposed to high concentration of the nuclease by one or more techniques, thus generating a list of candidate off-target sites, which are also analyzed comparatively with the bioinformatic predictions. Finally, the top ranked off-target sites are interrogated by deep sequencing for targeting by the nuclease in the selected target cells in conditions best representative of the intended clinical protocol. These studies should be conducted with proper positive and negative controls to determine sensitivity thresholds. Standard or threshold acceptance values for off targets are hard to provide across platforms, target cells and applications, and should be determined accordingly to the intended use.
Large genomic alterations, deletions and translocations are also induced, albeit to lower extent than NHEJ and HDR-mediated repair, at the DNA DSB sites, and are all difficult to evaluate. This is particularly true for allele drop-outs due to large deletions, which can encompass large segments of DNA. These events may be of particular concern if they lead to hemizygosity or even homozygosity for a loss-of-function mutation in a tumor suppressor gene. The possible contribution to loss of heterozygosity by gene conversion in the course of repair of a DNA DSB should also be considered. Efforts should go into ruling out the occurrence of unwanted on-target genomic alterations above a threshold limit of detection and/or expectation. Moreover, the possible occurrence of genomic rearrangement involving sensitive loci should be cause for discarding the candidate reagents. When addressing the overall safety of a cell product that may comprise a small fraction of cells bearing genomic alterations below the threshold of detection one may be able to draw upon available past experience with gene- and cell-based interventions using the same or other platforms with the same target cells.
Biodistribution studies of genome edited cells in suitable xenogeneic immunocompromised recipients should be performed to establish comparable behavior to untreated cells. Targeted editing by nucleases may leave a genetic scar. Such scars may be traceable, depending on the mechanism of repair of the DNA DSB. NHEJ-mediated DSB repair usually introduces small nucleotide insertion/deletions (indels) at the target site, which can be identified by deep sequencing the locus. However, some editing events might be missed because the original sequence is reconstituted or has been lost by a large deletion or because it was involved in a translocation. If only one base has been changed, it will be difficult to distinguish it from a sequencing error. Homology directed repair (HDR) of DSB can more easily be tracked because of the templated sequence changes in the target locus. Whenever feasible, strategies should be adopted to allow reliable tracking of the edited cells, for instance by recoding part of the target sequence in the template to introduce a traceable genetic marker. These base changes might also serve to protect the template from the action of the nuclease and improve the efficiency of editing. The genetic modifications introduced during editing could be used to track the fate, survival and biodistribution of the edited cells and their progeny. These studies will help to establish safety and efficacy of the treatment and address the possible relationship of eventual adverse events with the editing process (i.e. to distinguish the possible origin of abnormal differentiation, growth or transformation of some edited cells vs. background disease or age-related events). However, some edited cells may still escape tracking. Tracking of cells edited by base editors or epigenetic editors (see below) may prove even more difficult or perhaps impossible.
DNA DSB might induce DNA damage response in a dose-dependent manner as well as other signaling and transcriptional responses in cells treated for editing. P53-mediated responses have the potential to induce cell senescence with long-term detrimental effects and selection of p53+/- or -/- variants. The occurrence, extent and specific modes of such responses to genome editing still need to be investigated in most target cells and applications. Furthermore, combination of DNA DSB with vectors used to deliver the repair template for HDR may induce cumulative activation of innate immune sensors and trigger more detrimental responses. Such responses might only have transient effects but if robust and prolonged they might impact the cell survival, extent and time to engraftment, clonal composition and long-term stability of an engineered cell or tissue graft.
There are continuously emerging new technology platforms which introduce new editing modalities with broader reach and potentially improved precision and safety. For example: Break-less editors, Base editors, Prime editors (Anzalone et al., 2020). These new strategies are expected to provide improved editing precision at the target site by diminishing the spectrum of potential outcomes and to alleviate the burden imposed on the target cell by the DNA DSB. However, these new strategies also raise specific issues concerning monitoring for off-target effects. Specific tests might need to be designed to address genome wide specificity of these editors. In particular, many of these editors exploit the editing domain of an enzyme with constitutive activity independent of the binding of the fusion protein to DNA. Thus, off-target activity might be displayed semi-randomly in the genome and thus independently from the nearby occurrence of homology to the intended target sequence. Because of its semi-random occurrence such off-target activity may escape detection when investigating bulk treated cells, where semi-randomly distributed rare events would become noise. A possible strategy to address this issue is to compare SNVs among several single cell-derived clones from the treated cells.
In vivo genome editing still remains challenging because it requires an effective and safe delivery of the editing machinery to sufficient numbers of the relevant cell type. Current platforms support either stable high-level expression of editors with concomitant increased risk of toxicity, off-target activity and immunogenicity (such as when using AAV vectors) or they fail to achieve satisfactory efficacy due to low expression level. Nanoparticle based delivery methods represent a promising approach for short-term expression, but are still difficult to target to tissues other than the liver. Furthermore, most genome editors comprise at least some components of bacterial origin and are thus likely to be immunogenic. The sustained expression or even the residual presence of such material in the edited cells might impact their survival in vivo and this risk should be appropriately investigated before clinical testing.
Appendix 6. Informed Consent Standard for Stem Cell-Based Interventions Offered Outside of Formal Clinical Trials
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Download a PDF of ISSCR Informed Consent Standards for Stem Cell-Based Interventions, Version 1.0, 12 August 2019
Return to TOC | 2021 Guidelines for Stem Cell Research and Clinical Translation