Germline Gene Therapy Research Articles

Germline Gene Therapy to Prevent Transmission of Inherited Mitochondrial Diseases

Germline Gene Therapy to Prevent Transmission of Inherited Mitochondrial Diseases

O-044 The promise and peril of germline gene therapy

O-044 The promise and peril of germline gene therapy

Evaluation of guide-free Cas9-induced genomic damage and transcriptome changes in pig embryos

Cas9 protein without sgRNAs can induce genomic damage at the cellular level invitro. However, whether the detrimental effects occur in embryos after Cas9 treatment remains unknown. Here, using pig embryos as subjects, we observed that Cas9 protein transcribed from injected Cas9 mRNA can persist until at least the blastocyst stage. Cas9 protein alone can induce genome damage in preimplantation embryos, represented by the increased number of phosphorylated histone H2AX foci on the chromatin fiber, which led to apoptosis and decreased cell number of blastocysts. In addition, single-blastocyst RNA sequencing confirmed that Cas9 protein without sgRNAs can cause changes in the blastocyst transcriptome, depressing embryo development signal pathways, such as cell cycle, metabolism, and cellular communication-related signal pathways, while activating apoptosis and necroptosis signal pathways, which together resulted in impaired preimplantation embryonic development. These results indicated that attention should be given to the detrimental effects caused by the Cas9protein when using CRISPR-Cas9 for germline genome editing, especially for the targeted correction of human pathological mutations using germline gene therapy.

Ethical Challenges and Controversies in the Practice and Advancement of Gene Therapy

One of the most important technologies in modern medicine is gene therapy, which allows therapeutic genes to be introduced into cells of the body. The approach involves genetics and recombinant DNA techniques that allow manipulating vectors for delivery of exogenous material to target cells. The efficacy and safety of the delivery system are a key step towards the success of gene therapy. Somatic cell gene therapy is the easiest in terms of technology and the least problematic in terms of ethics. Although genetic manipulation of germline cells at the gene level has the potential to permanently eradicate certain hereditary disorders, major ethical issues such as eugenics, enhancement, mosaicism, and the transmission of undesirable traits or side effects to patients’ descendants currently stymie its development, leaving only somatic gene therapy in the works. However, moral, social, and ethical arguments do not imply that germline gene therapy should be banned forever. This review discusses in detail the current challenges surrounding the practice of gene therapy, focusing on the moral arguments and scientific claims that affect the advancement of the technology. The review also suggests precautionary principles as a means to navigate ethical uncertainties.

Gene therapy – Principles and Applications

The concept of gene therapy dates back to the 1960s and 1970s and it is yet to come of age, as there is a paucity of reliable, long-term data on the safety and efficacy of this novel therapy. It is fast emerging as a promising approach to treat, cure or prevent genetic based diseases by correction of defective genes that are responsible for disease development. Gene therapy is a key treatment strategy for disorders caused by a missing or faulty gene. It involves introduction of functional genes into appropriate cells so that they can produce sufficient amounts of proteins, encoded by the transferred gene or turn off genes that are causing problems and eventually resulting in permanent correction of the disorder. While the concept of gene replacement therapy is mostly suitable for recessive diseases, novel strategies have been suggested that are capable of also treating conditions with a dominant pattern of inheritance. Gene therapy treatments are of broadly two types, Germline gene therapy, & Somatic Germline gene therapy. There are two methods for administering gene therapy -Ex vivo & in-vivo. There are two main parts to gene replacement: Genes and Vectors. Gene therapy is useful for hematologic conditions as Hemoglobinopathies, Hemophilia and Inherited bone marrow failure. Future Prospective of Gene Therapy in Transfusion Medicine, work is being done to explore ex vivo generation of RBCs utilizing various cell fractions, including hematopeitic progenitor cells from cord blood and adult sources. Cost of therapy and ethical issues are the limiting factors for the use of Gene therapy.

Male Perspective of Infertility and Germ line Gene Therapy: An Islamic and Medical Perusal

Male Perspective of Infertility and Germ line Gene Therapy: An Islamic and Medical Perusal

Restoration of RPGR expression in vivo using CRISPR/Cas9 gene editing

Mutations in the gene for Retinitis Pigmentosa GTPase Regulator (RPGR) cause the X-linked form of inherited retinal degeneration, and the majority are frameshift mutations in a highly repetitive, purine-rich region of RPGR known as the OFR15 exon. Truncation of the reading frame in this terminal exon ablates the functionally important C-terminal domain. We hypothesized that targeted excision in ORF15 by CRISPR/Cas9 and the ensuing repair by non-homologous end joining could restore RPGR reading frame in a portion of mutant photoreceptors thereby correcting gene function in vivo. We tested this hypothesis in the rd9 mouse, a naturally occurring mutant line that carries a frameshift mutation in RPGRORF15, through a combination of germline and somatic gene therapy approaches. In germline gene-edited rd9 mice, probing with RPGR domain-specific antibodies demonstrated expression of full length RPGRORF15 protein. Hallmark features of RPGR mutation-associated early disease phenotypes, such as mislocalization of cone opsins, were no longer present. Subretinal injections of the same guide RNA (sgRNA) carried in AAV sgRNA and SpCas9 expression vectors restored reading frame of RPGRORF15 in a subpopulation of cells with broad distribution throughout the retina, confirming successful correction of the mutation. These data suggest that a simplified form of genome editing mediated by CRISPR, as described here, could be further developed to repair RPGRORF15 mutations in vivo.

Commentary on MJM Crossroads: Human Germline Gene Therapy

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SURVEY ON PERCEPTION OF GERMLINE GENE THERAPY AMONG JAPANESE MEDICAL STUDENT

Germline gene therapy (GGT) has potential to be radical treatment approach for monogenic genetic disorder. Because recent CRISPR technology enables us to edit human genome with high efficacy and lowest off-targeting, technical issues for GGT have been almost solved. Mitochondrial replacement therapy (MRT) is also only radical germline gene therapy for heritable mitochondrial disease. Japanese government has issued a statement prohibiting GGT, including MRT, and emphasizing the need for discussions in a wide range of perspectives.

Gene therapy: a double-edged sword with great powers.

Gene therapy is the treatment of a disease through transferring genetic material into cells of the patients. In the recent several years, gene therapy has experienced rapid progress and achieved huge success. Over two dozens of gene therapies have been approved for clinical use by the drug regulatory agencies from different countries. However, concerns about its efficacy and safety have accompanied gene therapy since its birth. In the present manuscript, we first introduce various strategies employed in gene therapy, which includes ex vivo gene delivery v.s. in vivo gene delivery; gene addition v.s. genome editing; inherited disease v.s. acquired disease; and somatic gene therapy v.s. germline gene therapy. Then we discuss the clinical outcomes of some approved gene therapies. We finish our discussion with the safety issues related to gene therapy. We will see that with the technology improvement, somatic gene therapy has been proved to be efficient and safe enough for clinical practice. However, germline gene therapy has important efficiency and safety issues at present, and should not be put into clinical practice before these issues are solved.

Ethical and Legal Perspective on Germ-line Gene therapy

When humans discovered the code of life- DNA (deoxyribonucleic acid), a complex molecule that guides the growth, development function and reproduction of everything alive, new frontiers of possibilities opened to us. Information is encoded in the structure of this molecule. Four nucleotides are paired and make up a code that carries instructions. As soon as DNA was discovered, people tried to tinker with it. In the 1990s, there was a brief foray into human engineering. To treat maternal infertility, babies were made to carry genetic information from three humans making them the first humans ever to have three genetic parents. Today, there are super muscled pigs, fast growing salmon, featherless chickens and see through frogs. On the fun side, there are also glow in the dark Fluorescent Zebra fish available for as little as ten dollars in the US. This is impressive but until recently, gene editing was expensive, complicated and took a long time to do. This has now changed with a revolutionary new technology, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats). Overnight the cost of engineering shrunk by 99%. Instead of a year, it takes few weeks to conduct an experiment and basically, everybody with a lab can do it. It’s hard to get across how big of a technical revolution CRISPR is. It literally has the potential to change humanity forever, for example; ending disease, designer babies, eternal youth along with super strength, undefeatable army etc. Scenarios like these are far off into the future if they ever become possible at all but they are not impossible. While this may be a tempting reason to ban genetic engineering, it would certainly be a mistake. Banning human genetic engineering would only lead science wandering off to a place and jurisdiction we’re uncomfortable with. Only by responsibly participating can we make sure that further research is guided by precaution, reason, oversight and transparency. By gaining a legal insight into the ramifications of such rapid scientific and technological developments and its potential, we as a society will be able to better secure and control it in alignment with legal understanding. Law provides a torch that sheds ideal light on several aspects and nuances that come with the application of modified knowledge in the field of biotechnology, both internationally and nationally with the aim to prevent its misuse and protect and promote the rights of an individual as well as the collective society. And therefore, this thesis aims to explore the various angles of the relationship of biotechnology (germ-line gene therapy) with law in several areas. Two minutes of thought would lead to recognition of myriad issues that one may experience in harmonizing such advances and adjusting them on the lives of individuals and productively using them as a society without breaching unauthorized territory. Ethical, moral, religious and legal perspectives have been covered in the following chapters.

Principles of and strategies for germline gene therapy.

Monogenic disorders occur at a high frequency in human populations and are commonly inherited through the germline. Unfortunately, once the mutation has been transmitted to a child, only limited treatment options are available in most cases. However, means of correcting disease-causing nuclear and mitochondrial DNA mutations in gametes or preimplantation embryos have now been developed and are commonly referred to as germline gene therapy (GGT). We will discuss these novel strategies and provide a path forward for safe, high-efficiency GGT that may provide a promising new paradigm for preventing the passage of deleterious genes from parent to child.

Mitochondrial genetic medicine.

Inherited mitochondrial DNA (mtDNA) diseases were discovered 30 years ago, and their characterization has provided a new perspective on the etiology of the common metabolic and degenerative diseases, cancer, and aging. The maternally inherited mtDNA contains 37 critical bioenergetic genes that are present in hundreds of copies per cell, but the 'mitochondrial genome' encompasses an additional 1,000-2,000 nuclear DNA (nDNA) mitochondrial genes. The interaction between these two mitochondrial genetic systems provides explanations for phenomena such as the non-Mendelian transmission of the common 'complex' diseases, age-related disease risk and progression, variable penetrance and expressivity, and gene-environment interactions. Thus, mtDNA genetics contributes to the quantitative and environmental components of human genetics that cannot be explained by Mendelian genetics. Because mtDNA is maternally inherited and cytoplasmic, it has fostered the first germline gene therapy, nuclear transplantation. However, effective interventions are still lacking for existing patients with mitochondrial dysfunction.

Patients Suffer While the Science Establishment Resists Innovative Therapies

Human gene therapy has been up to now of a type that affects only the patient being treated; it has not modified sperm or eggs cells or embryos in a way that would constitute “germ line gene therapy” (GLGT) by creating a heritable change and affecting future generations. Preclinical research has progressed almost to the point where GLGT interventions will be possible with a reasonable likelihood of success, but such clinical trials are currently prohibited: NIH's Recombinant DNA Advisory Committee is not permitted even to consider such proposals, and the FDA cannot use appropriated funds to review such trials. Such absolute prohibitions are bad for patients and bad public policy.

I-GONAD: a robust method for in situ germline genome engineering using CRISPR nucleases

We present a robust method called improved-Genome editing via Oviductal Nucleic Acids Delivery (i-GONAD) that delivers CRISPR ribonucleoproteins to E0.7 embryos via in situ electroporation. The method generates mouse models containing single-base changes, kilobase-sized deletions, and knock-ins. The efficiency of i-GONAD is comparable to that of traditional microinjection methods, which rely on ex vivo handling of zygotes and require recipient animals for embryo transfer. In contrast, i-GONAD avoids these technically difficult steps, and it can be performed at any laboratory with simple equipment and technical expertise. Further, i-GONAD-treated females retain reproductive function, suggesting future use of the method for germline gene therapy.

The Council of Europe should not reaffirm the ban on germline genome editing in humans.

In reaction to recent progress in human embryo genome editing, the Council of Europe is resolutely on its way to reaffirming a 20‐year‐old policy banning all inheritable interventions involving the human genome. This approach, which was well justified two decades ago, is now outdated, overly restrictive and will hamper promising research for germline gene therapy. A ban no longer makes sense from an ethical point of view. Until recently, there has been a worldwide consensus among scientists and policymakers not to modify the human germline. The main rationale was that classical genetic engineering technologies in humans were inefficient and imprecise. The risks were simply too great. But the risk assessment was based on recombinant DNA technology, which is much less precise than the new genome editing technologies—with the flagship CRISPR/Cas9 system—that are much more efficient and precise. Although they are not yet sufficiently safe to be used in clinical trials, research has made rapid progress in improving efficiency and precision of the CRISPR technology [1]. With further improvements, gene‐editing technology therefore has …

It’s In Your Genes: Recent Considerations in Germline versus Somatic Gene Therapy

It’s In Your Genes: Recent Considerations in Germline versus Somatic Gene Therapy

CRISPR/Cas9-mediated somatic and germline gene correction to restore hemostasis in hemophilia B mice.

Hemophilia B (HB) is an X-linked disorder caused by defects of F9 encoded coagulation factor IX, which is an ideal model for gene therapy. Most existing HB gene therapies are based on viral mediated gene supplementation, which could increase immunoreaction. In this study, CRISPR/Cas9 system was used for gene correction in an F9 mutant HB mouse model in both adult mice (in vivo) and in germline cells (ex vivo). In vivo, naked Cas9-sgRNA plasmid and donor DNA were delivered to HB mice livers to recover the mutation via hydrodynamic tail vein (HTV) injection. 62.5% of the HTV-treated mice showed a detectable gene correction (>1%) in the F9 alleles of hepatocytes, which was sufficient to remit the coagulation deficiency. Ex vivo, three different forms of Cas9 were microinjected into germline cells of HB mice to investigate their efficiency and safety in gene correction. Cas9 protein showed higher gene recovery rates, less embryo toxicity, and lower mosaic repair percentage, making it more suitable for germline gene therapy. Our study strongly supports that CRISPR/Cas9-mediated genome editing is feasible in gene therapy of genetic disorders.

CRISPR-Cas9, germinal cells and human embryo

The performance of the molecular tool using CRISPR-Cas9, which makes it possible to induce targeted modifications of the DNA, has found numerous applications in research and open promising prospects in human clinic. CRISPR-Cas9 has been widely used to generate transgenic animals after targeted modification of the genome at the zygotic stage. It was also tested on human embryos on an experimental basis. Although there are potential medical indications that may justify a targeted modification of the embryo or germ cell genome, the uncertainties regarding the efficacy and safety of the method do not allow us to consider implementing such germline gene therapy in the short-term. However, it is necessary to weigh the scientific and ethical issues involved in this approach.

Leber Hereditary Optic Neuropathy: Exemplar of an mtDNA Disease.

The report in 1988 that Leber Hereditary Optic Neuropathy (LHON) was the product of mitochondrial DNA (mtDNA) mutations provided the first demonstration of the clinical relevance of inherited mtDNA variation. From LHON studies, the medical importance was demonstrated for the mtDNA showing its coding for the most important energy genes, its maternal inheritance, its high mutation rate, its presence in hundreds to thousands of copies per cell, its quantitatively segregation of biallelic genotypes during both mitosis and meiosis, its preferential effect on the most energetic tissues including the eye and brain, its wide range of functional polymorphisms that predispose to common diseases, and its accumulation of mutations within somatic tissues providing the aging clock. These features of mtDNA genetics, in combination with the genetics of the 1-2000 nuclear DNA (nDNA) coded mitochondrial genes, is not only explaining the genetics of LHON but also providing a model for understanding the complexity of many common diseases. With the maturation of LHON biology and genetics, novel animal models for complex disease have been developed and new therapeutic targets and strategies envisioned, both pharmacological and genetic. Multiple somatic gene therapy approaches are being developed for LHON which are applicable to other mtDNA diseases. Moreover, the unique cytoplasmic genetics of the mtDNA has permitted the first successful human germline gene therapy via spindle nDNA transfer from mtDNA mutant oocytes to enucleated normal mtDNA oocytes. Such LHON lessons are actively being applied to common ophthalmological diseases like glaucoma and neurological diseases like Parkinsonism.