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If on-target editing frequencies of clinically relevant cell types are high enough to be therapeutically useful, genome editing may eventually outperform gene replacement traditional gene therapy in terms of safety, provided that off-target changes do not pose similar risks by modifying genes associated with cancer. Another potential broad application of genome editing is precisely targeted integration of a gene expression cassette into a so-called safe genomic harbor, chosen because it is conducive to robust transgene expression and allows a safe insertion that does not have a detrimental effect on adjacent genes.

This approach may ensure predictable and robust expression of a therapeutic gene without the risk of oncogenesis caused by inadvertent insertional activation of an oncogene. Targeted integration into a safe harbor and in situ correction of mutations are both potentially widely applicable to stem cell—based therapies as long as the targeted cells are amenable to extensive in vitro culture selection and expansion prior to clinical use. One can envisage increasing application of these types of genome editing as the ability to grow and differentiate different types of cells in culture improves, particularly in conjunction with differentiation from pluripotent cells Hockemeyer and Jaenisch, A unique application of genome editing relative to standard gene therapy strategies is targeted gene disruption.

Indeed, clinical testing of gene disruption using zinc finger nucleases ZFNs is already under way, with some indication of benefit for T-cells Tebas et al. Multiple nuclease platforms have been developed or improved in the past years, making it likely that additional such platforms will be developed in the near future. These issues are not new, however, nor are they specific to the CRISPR-Cas9 system; many of them have already been confronted and addressed in the context of earlier gene therapy and genome-editing applications.

See also Chapter 3. Importantly, while genome editing by NHEJ is precisely located by where the DNA break or nick is produced, it is not possible to predict the size or sequence of the resulting change in a single cell or the variability of the changes indels among a group of cells.

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In genome editing by HDR, a DNA template is used either to create one or more nucleotide changes, perhaps to match a known human reference sequence, or to insert a novel sequence e. Three of these trials have been completed, one is ongoing, and two are currently recruiting participants. In contrast to NHEJ, HDR-mediated genome editing allows scientists to predict both where the edit will occur and the size and sequence of the resulting change.

The intent of each of these modifications could be to treat or prevent a disease but could also be to modify or, in principle, even create novel phenotypic traits in the treated cells or tissues.

Cell cell hybridization or somatic cell hybridization

It is important to note, for example, that one can use genome editing to achieve enhancement of a cellular property e. Such cellular enhancement with intent to modify disease course needs to be distinguished from the concept of enhancement aimed at creating a desired or novel organismal feature in humans a topic discussed in detail in Chapter 6.

Table provides examples of the types of human diseases that might. Even though this list is not comprehensive, it highlights the broad range of potential applications.

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A more subtle use of genome editing to correct a disease-causing variant is to insert the wild-type DNA copy of the mRNA complementary or cDNA into an endogenous locus to correct downstream mutations Genovese et al. Concerning the liver as a target organ, it has been shown that targeted insertion of a clotting factor transgene downstream of the promoter of the albumin gene in a fraction of hepatocytes may rescue the hemophilia bleeding phenotype in mouse models Anguela et al. Several potential applications of genome editing entail causing gene disruption, provided that the delivery of the nuclease does not lead to loss of the treated cells because of toxicity or immune rejection.

In T-cell immunotherapy, a promising application of genome editing is single or multiplex disruption of genes that may antagonize, counteract, or inhibit the activity of exogenous cell-surface receptors introduced into T-cells to direct them against tumor-associated antigens Qasim et al. These strategies can strongly potentiate current cell-based immunotherapy strategies, possibly overcoming current barriers that limit efficacy in most solid tumors.

All types of genome editing involve consideration of certain parameters that together determine the efficacy and potential toxicity of a genome-. These scientific and technical considerations inform how and why a particular approach is chosen to meet a research or therapeutic goal; they also impact the nature of the data that will be available for the regulatory evaluations that will be required for potential preclinical testing, clinical trials, review, and ongoing oversight of these methods. The choice of nuclease includes the platform type, which can be based on protein-DNA recognition e.

When developing protein-based DNA-binding domains that are made using zinc fingers and TAL effectors, extensive engineering and improvement are possible for each specific sequence-binding domain, such that it is difficult to make a general prediction on the performance and specificity of the overall platform. That is, for ZFNs and TALENs, optimization of performance activity and specificity often requires work for each nuclease that may or may not translate to another nuclease.

In contrast, when RNA-based nucleases such as Cas9 are developed, general improvements are made to the platform itself and should translate to each specific target sequence. This fact has implications for the ease or speed with which genome-editing systems designed for one clinical application could be adapted to target others.

Genome editing can be carried out ex vivo or in vivo. In ex vivo editing, it is possible to conduct a number of checks on the edited cells before they are administered to a patient because the cells are first manipulated in the laboratory. Ex vivo editing, which occurs outside the body, is suitable only for certain cell types, however. Ex vivo genome editing can be performed by isolating and manipulating a population of the intended target cells outside the body and then transplanting those cells into an individual.

The source of cells can be. Whether the cells are sourced from the same patient or a matched donor, the administered cells often have stem cell—like properties, which may allow their self-renewal and long-term maintenance in vivo, as well as repopulation of the treated tissue with their genetically modified progeny. In some approaches, the cells can be treated in culture to induce commitment or differentiation toward a desired cell type or lineage before being administered to the patient.

Otherwise, the edited cells can be differentiated somatic cells, such as short-lived or long-lived immune effector cells that are expanded and genetically modified ex vivo to enhance their activity against a tumor or infectious agent. This list likely will grow as scientific knowledge and techniques improve.

An expanded repertoire of cell types has the potential to increase the range of possible ex vivo genome-editing applications.

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If HDR is intended, a homologous template is also required. Targets of in vivo genome editing may include long-lived tissue-specific cells, such as muscle fibers, liver hepatocytes, neurons of the central nervous system, or photoreceptors in the retina, but may also include rare, tissue-specific stem cells and other types of cells that cannot easily be harvested and transplanted. Relative to ex vivo approaches, however, in vivo approaches pose greater challenges with respect to efficient delivery of the genome-editing machinery to the right cells in the body, ensuring that the correct location in the genome has been successfully edited, and minimizing errors resulting from off-target editing.

A number of additional scientific and technical considerations related to both ex vivo and in vivo genome editing inform the development of human genome-editing systems. To carry out ex vivo genome editing, it is necessary first to isolate the relevant cell types from an appropriate tissue source or to generate them from pluripotent stem cells, and then to grow and modify them ex vivo and. There are several advantages to the ex vivo strategy: only the intended cells are exposed to the editing reagents, there is a wide choice of delivery platforms that can best be fitted to each cell type and application, and it is possible to characterize and even purify and expand the edited cells before administration.

Currently this process has been established for only a few cell types, including cells that will eventually give rise to skin, bone, muscle, blood, and neurons. The range of possible ex vivo genome-editing applications will expand with the development of scientific knowledge about how to isolate additional primary cell types and derive other cell types from pluripotent cells, grow the cells ex vivo, and ultimately transplant them back into patients successfully and safely.

Ex vivo genome-editing strategies have a number of expected limitations, which are common to all attempts at culturing cells ex vivo. These limitations include the need for prolonged culture and expansion from a few cells or even a single founder cell, both of which entail the risk of accumulating mutations, as well as incurring replicative exhaustion. This issue is particularly relevant for genome editing because inducing double stranded breaks in DNA, as is required to initiate the process, may itself trigger such cellular responses as apoptosis cell death , differentiation changing cell type , cell senescence aging , and replicative arrest cells stop dividing.

All of these cellular responses are detrimental to cell expansion and maintenance of pluripotency. These limitations represent significant hurdles to ex vivo genome editing because most therapeutic applications require substantial numbers of cells for infusion. Overcoming these hurdles will require better ways to culture cells, better understanding of the safety risks associated with genomic accrual of random mutations in these settings, and reliable assays for assessing such events.

Additional hurdles are the ability to fully control the commitment and differentiation of cells in culture and their purification from the source pluripotent cells. This is an important consideration because administration of immature cells may be associated with a risk of tumorigenesis or failure to integrate functionally within the tissue. Despite these limitations, ex vivo genome editing has the advantage that cells with the desired alteration can be selected and the accuracy of the alterations validated before transplantation to the patient.

Additional considerations for in vivo genome editing are linked to the choice of the delivery platform for the editing machinery because this choice impacts the extent, time course, and in vivo biodistribution of the genome-editing tool. This consideration has major implications for poten-. Efficient editing of the intended genomic site usually requires a high level of intracellular nuclease expression, even though this often can be for only a short time to prevent excess toxicity and off-target activity.

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  8. Whereas short-term, high expression of the genome-editing nuclease can be obtained relatively easily for cells cultured in vitro, it is more challenging in vivo. Finally, in an in vivo setting there could be unintentional inadvertent modification of the germ cells or primordial germ cells; therefore, preclinical development of in vivo editing should address the risk of modification of germ cells resulting in heritable changes that could be passed on to future generations and minimize this potential risk in humans enrolled in clinical trials.

    In general, the risk of germline transmission associated with the administration of ex vivo genome-edited cells is likely to be low if one can show that the editing reagents do not remain associated with the treated cells and are not shed in active form at the time of administration. In these conditions, nonclinical studies of germline transmission may not be necessary. On the other hand, in vivo administration of editing reagents would require assessment of their potential biodistribution to the gonads and activity on germ cell genomes.

    These parameters will be strongly influenced by the delivery platforms used and the timing and route of delivery. When viral vectors are used to deliver the nuclease, the preclinical studies might take into consideration accumulating knowledge from animal and human studies concerning the potential of these vectors to reach germline cells. A suggested approach to studying the potential of germline transmission in such nonclinical models would be to follow a decision tree, in which a positive finding triggers the next level of investigation.

    Molecular assays could be designed to track the occurrence of indels at the intended or surrogate nuclease target sites, provided that such sites exist in the genome of the species used for the study with sufficient affinity for the nuclease to support the sensitivity of the assay. Many limitations exist when conducting such studies in surrogate animal species, as already discovered for several gene therapy products, including the low sensitivity of the available assays, species-specific differences in vehicle biodistribution and access to the gonadal cells, and the general difficulties of testing transmission to.

    Testing of semen can be done at various points during this time interval; if samples are positive, the testing should continue, and the respective regulatory authorities should be notified.

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    On the other hand, there are currently no noninvasive means of monitoring women for germline transmission. In vivo delivery of proteins and nucleic acids is currently done with either of two types of platforms. This approach can expose therapeutically irrelevant cell types in the patient to the potential toxicity of the nuclease. The second type of platform relies on viral vectors that can provide robust and tissue-specific expression, but they also are frequently long-lived and more likely to provoke an immune response. Self-complementary rAAV8 vectors scAAV , for example, have been shown to mediate continued expression of the engineered nuclease.

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    Moreover, all current formulations of editing machinery contain elements that are derived from proteins of common microbial pathogens, which could trigger primary or secondary immune responses in treated individuals. As has been well documented in viral gene therapy studies, immune recognition of viral vector proteins may lead to rapid and complete clearance of cells that have received the editing machinery, which eliminates the benefit of the treatment. The risk of clearance of the edited cells is exacerbated by preexisting immunity and by the extent and duration of expression of the antigen.

    Another major hurdle for both ex vivo and in vivo editing is that targeted insertion of a DNA sequence into postmitotic cells, such as neurons, is not. In contrast, NHEJ, which is active in nondividing cells, has been harnessed mainly for the generation of indels to inactivate a gene.

    However, NHEJ can, with modifications to the methods, be used to generate site-specific gene insertions e.

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    Most recently, it was reported that one of these methods, homology-independent targeted gene integration, or HITI, allows targeted knock-in of DNA sequences in dividing cells e. In vivo genome editing is a highly sought-after application that has been shown to be feasible and potentially therapeutic in some mouse models. Substantial challenges to its translation to the clinic remain, however, at least in the current modalities of administration.