September 01, 2016

Science in the World of Gene Editing and Gene Therapy

For much of the last century, the choice of a therapeutic for many diseases has been to use drugs selected on the basis of the predicted response of the majority of patients to that particular drug. Although this “one drug fits all” paradigm has been a mainstay of medical practice, over the years it has been demonstrated that many patients with the same disease respond differently to various common drug regimens and that the genetic makeup of the individual patient likely plays a significant role in these variable drug responses.1

It has long been suggested that the better method to treat patients would be to match the patient to the drug rather than the drug to the patient. There have been many attempts to personalize treatments to fit the individual patient, but the necessary tools and information needed to go forward with a true personalized-medicine approach were lacking until the completion of the Human Genome Project (HGP) in 2003. The HGP was a global endeavor conducted with the goal of identifying the entire DNA sequence of the human genome, including chromosomes, genes, and noncoding areas. Noncoding is a misnomer, as these areas of the genome are important for the regulation of genes and gene expression.

The original published HGP “map” was just a beginning, and since then there has been a great deal of additional DNA sequencing, genome studies, and bioinformatics research to further identify the locations and roles of each of the approximately 20,000+ human genes identified to date. A major goal of this research is to be able to use the information to help improve diagnosis and treatment of diseases, as well as to identify genetic predisposition to disease, genetic errors associated with particular diseases, and genes and/or defective DNA sequences for developing treatments using gene editing.

The HGP and the subsequent sequencing research should assist medical science to more quickly move from the “one drug fits all” model to a more precise ability to diagnose and treat diseases in the individual. This is a premise behind the push toward precision medicine. Although the idea of precision medicine is as old as medicine itself, the current concept of precision medicine is a model of customization or “individualization” of health care tailored to the individual as well as to the specific disease being treated.2 Genetic testing is already being used to some extent in diagnostics; however, as precision medicine matures, patients’ genetics will become more and more important in their clinical workup, diagnosis, and treatment.

A major premise of the precision medicine paradigm is that a significant component of human health and disease is based on genetics, either hereditary or acquired. With advancements in genomic research and sequencing technologies, scientists are beginning to uncover genetic components of many diseases. However, there remain many barriers to the use of this information for therapeutic intervention, one of which has been our limited ability to alter a person’s genome. This article will briefly discuss the history and current status of gene editing and gene therapy, with particular emphasis on the recent discovery and use of the CRISPR/Cas9 gene editing system.

A Brief History of Gene Therapy

The discovery of retroviruses and subsequent demonstration of their ability to randomly insert foreign DNA into a genome led to their use as a delivery system for early gene therapy. In 1990, using this system, the first gene therapy trial was initiated, targeting an inherited, severe immunodeficiency syndrome called ADA-SCID.3 Unfortunately, five of the 20 patients treated developed leukemia, which raised significant safety concerns regarding the use of retroviral vectors for delivery of the genetic material. The adverse findings, along with a growing public fear of gene therapy and genetic engineering, slowed both funding and progress in this field.

A concern with the use of retroviral delivery systems was that they can insert new (exogenous) DNA randomly into the target genome, which can lead to cancerous transformations. Therefore, investigators began to look for alternate ways to “fix” the defective DNA rather than replacing it. For years, scientists had been exploiting homologous recombination (exchanging two similar or identical molecules of DNA) in order to introduce DNA changes or repairs in the genomes of targeted organisms.4 This type of DNA repair mechanism is also a naturally occurring cellular process used to create genetic variation during reproduction and to repair defective/mutated DNA during cell division. The efficiency of DNA recombination is extremely low, but was found to be significantly increased if the DNA at the target site was first damaged or cut, using site-directed endonucleases. Endonucleases (or nucleases) are enzymes that can cut specific DNA sequences within the genetic material of a cell. They include: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system.

Meganucleases (homing endonucleases) are able to recognize and cut specific DNA sequences, resulting in a double-stranded DNA break at the target site. Many different naturally occurring meganucleases have been discovered, and other genetically engineered meganucleases have been developed to target multiple genomic DNA sites; however, the engineering process is very difficult and costly.

In the early 2000s, investigators developed the more flexible ZFN system.5 ZFNs can also be designed to target and cut a specific DNA sequence within the genome, and they were soon adapted for use in genome editing of human, animal, and plant cells.6 Each of the ZF DNA-binding domains must be reengineered for each newly targeted gene or targeted DNA sequence, but they do not always have the needed predicted DNA sequence specificity for targeting. Again, engineering and producing ZFNs is time-consuming and quite expensive.

A more recent discovery of a novel DNA recognition code, termed transcription activator-like effectors (TALEs), provided an even more flexible alternative to ZFN.7 There are only four TALE subunits, one for each DNA base (adenine, guanine, thymine, and cytosine), making the design of TALENs more straightforward and more effective than the earlier site-specific nucleases discussed.

Even more recently, another site-specific nuclease system was discovered: the clustered regularly interspaced short palindromic repeat-associated nuclease Cas9 system (CRISPR/Cas9). This new technology has proved to be a significant breakthrough for gene editing as well as a considerable “upgrade” over both ZFNs and TALENs technology.8

Unlike ZFNs and TALENs, the sequence specificity of the CRISPR/Cas9 system is not driven by a “programmable” string of protein subunits. Instead, an associated RNA molecule, called a short guide RNA (sgRNA), guides the Cas9 nuclease to the targeted gene or complementary DNA sequence. The CRISPR/Cas9 system is easier to use and less costly than the other endonuclease systems, and the RNA-based specificities abrogate the requirement for protein engineering.

CRISPR/Cas: Current Approaches and Future Directions

CRISPR has quickly become the gene editing tool of choice for genomic research, and most molecular and genetic laboratories in the world are now using CRISPR in their studies. With so many working with CRISPR, progress in the field of gene editing has been rapidly evolving, and new applications/technologies are being developed on a regular basis. These advances have led to an enormous amount of technical data on all aspects of CRISPR, including issues most critical for successful gene editing and gene therapies. These include: how to get CRISPR “products” into specific target cells (delivery); increasing its on-target cutting efficiency (efficacy); the ability to substitute a specific DNA sequence with another copy of the original DNA or insert a completely different sequence (gene knock-in) using homology-driven repair (specificity); and significantly reducing off-target DNA cleavage (unintended effects).

The first CRISPR experiments were performed on cell lines and mouse embryos using CRISPR-modified plasmids (circular DNA that can replicate gene transcription products without chromosomal involvement). Once inside the cell, plasmids begin producing Cas9 protein and sgRNA. When both are produced in the targeted cell(s), the sgRNA binds to the Cas9 protein, and the DNA sequence-specific component of the sgRNA searches for and binds with its complementary DNA sequence.9 For successful binding to the target DNA, the complex requires the presence of a specific DNA sequence (PAM) that immediately follows the DNA target site. The PAM site requirement limits the regions of the genome that are available for cleavage using the CRISPR/Cas9 system. Upon binding to the DNA target site, the Cas9 protein then cuts both strands of the chromosomal DNA, a process that again mimics naturally occurring DNA damage within a cell. The targeted cell responds to the damage by repairing the cut DNA through two competing processes: nonhomologous end joining (NHEJ), which “glues” the cut double-stranded pieces of DNA back together, or homology-directed repair (HDR), which uses a homologous DNA molecule (either an endogenous DNA template or an exogenous donor DNA template) to accurately repair the cut DNA (see fig. 1).

The delivery of Cas9 protein and sgRNA into cells using plasmids proved effective; however, many investigators were unable to achieve successful and/or specific editing using this approach.10 Another major problem with using plasmids is that the material can randomly integrate into the target cell’s genome, leading to abnormal cellular transformation or other unwanted/deleterious effects. Investigators soon discovered that delivery to the cell of a purified Cas9/sgRNA complex, termed ribonucleoproteins (RNPs), allowed for a more efficient gene knockout or repair. Also, delivery of the RNPs using methods such as direct transfection or electroporation removed the need to use a viral or a plasmid vector. The RNP method is currently the best available option for many cells, such as human blood cells, primary cells, and lymphocytes.11 The RNP approach works well for cells in culture, as well as for genetic engineering of germline cells (embryos and spermatic cells), and will likely be the mainstay for CRISPR in most areas of gene editing for the foreseeable future.

One of the primary clinical applications of CRISPR will be the genetic modification/repair of somatic (nongermline) cells, for example defective muscle cells in Duchenne muscular dystrophy and retinal cells in retinitis pigmentosa.12 For many of the somatic cell therapies, edited cells will be removed from the patient, treated/edited in culture outside the body (in vitro), and then reintroduced into the same patient. However for in vivo CRISPR-based human somatic cell therapies, there will need to be a mechanism capable of successfully delivering the Cas9 protein and sgRNA to the targeted tissue/cells. Direct injection of RNPs into the bloodstream is not currently a valid option, because the liver would remove most of the material without it ever reaching its target. One solution for in vivo delivery was to return to the use of a viral vector that can specifically “infect” the targeted organ or cells. Viral-based gene therapy approaches have already been approved in several clinical trials, and until nonviral alternative approaches can be perfected, they are likely to be the delivery mode in the majority of CRISPR gene editing trials.

After delivery, a next major concern with CRISPR is editing efficiency (the percentage of desired specific DNA cuts). The factors that govern efficient DNA cleavage and repair in cells are still somewhat unknown, and the efficiency of CRISPR has proven to be quite variable across various cell types. The RNP method appears to achieve consistently higher editing rates, but there still remains considerable variability in the cleavage efficiencies between guides targeting the same gene using RNP. In general, higher editing efficiencies are better; however, every genetic disorder is unique, so what fraction of cells will need to be “repaired” to alleviate the clinical manifestations of the targeted disorder remains another significant unknown. Newer Cas9 proteins have recently been discovered, which should allow future investigators to customize their Cas9 and sgRNA combination to find an efficient match for additional target areas within the genome.13

Following Cas9-mediated DNA cleavage, the cell repairs the induced lesion using either NHEJ or HDR (see fig. 1). NHEJ can often result in imprecise DNA repairs, while HDR is less prone to errors and can also be used to introduce or repair specific mutations near the cleavage site. Most genetic diseases that will be targets for CRISPR-based therapies will require HDR, and therefore many groups are focused on improving the delivery and efficiency of HDR. Similar to the delivery of Cas9 protein and sgRNA, there are multiple options for the delivery of the HDR template for repair.14

An issue with HDR is that it appears to be a repair mechanism specific only for dividing cells.15 Many of the targeted cells for gene therapy are not actively dividing (quiescent) or will never divide again (post-mitotic) and therefore use NHEJ in the normal DNA repair process. A potential solution is to modulate the cellular pathways responsible for HDR and NHEJ through the codelivery of CRISPR products and HDR proteins, or the delivery of CRISPR products along with some type of inhibitors of NHEJ. These strategies are still in their infancy and are likely years away from an effective product. One promising new candidate for increasing HDR efficiency is another RNA-directed endonuclease—Cpf1, which uses a shorter guide RNA than Cas9—that may allow for more cleavage and repair events and improve the frequency of HDR.16

Even in dividing cells the efficiency of HDR is low, and it is unclear whether the levels of HDR attainable will be sufficient for repair of defective cells in diseases like sickle cell anemia, cystic fibrosis, or other common genetic disorders.17

Most basis and clinical research on gene editing and repair are currently using the CRISPR system in their studies, but its use in genomic research has recently expanded from modifying genes to precisely cutting and repairing genomic DNA. Using a modified CRISPR/Cas9 system, scientists are now able to use it to directly regulate gene expression by directly activating or suppressing gene activity.18 These additional CRISPR applications will expand its use in exploring the genome, especially with regard to gene function at the cellular level.

Embryonic Gene Editing

Potential clinical applications of CRISPR (and other gene editing techniques) discussed so far have been presented in the context of the modification of genomes in somatic cells. However, gene editing research has also included numerous investigations on the effects of gene modifications in embryos and germline cells. The majority of the embryonic gene modification studies to date have been conducted in animals, but a few have involved the editing of human embryos. Several human embryonic gene modification studies are now in the early stages, and these will concentrate on exploring the role of various genes in early embryonic development (during the first few days post-fertilization).19

However there are two recent studies on human embryonic gene modification that have already been published where the goal (of both) was in part to explore the plausibility of using embryonic editing for therapeutic interventions. In the first of these studies, the authors attempted to modify embryonic genes known to be linked to Β-thalassemia (a genetically related blood disease).20 In the second, the investigators attempted to introduce specific gene mutations (already known to confer HIV resistance in adults) in the embryo with a goal of inducing genetic resistance to HIV infection.21 Both of these studies used nonviable human embryos. Each of the studies reported finding the presence of successfully edited cells as well as nonedited cells (mosaicism). Along with edited and nonedited cells, the first study reported finding a significant amount of off-target DNA cleavage. Although the authors of the second study did not find off-target effects, they indicated that undetected off-target modifications were probably present. In discussing their findings, both sets of investigators emphasized that there remain many very significant problems and issues with human embryonic gene editing that will need to be resolved before genetically modified embryos are safe enough to be implanted in humans. The safety issue here is extremely important because genes that are modified in the embryo (those that will be used for therapeutic applications) will have the potential of being passed on from one generation to all subsequent generations.

Although all of the studies on genetic modifications in human embryos to date have been carried out in the test tube, this is not the case in animal studies. Numerous studies have been published reporting on genetic modifications of animal embryos that have been subsequently implanted into surrogate mothers and taken to term. One such study used CRISPR to correct a disease-related genetic mutation in mouse embryos that resulted in the birth of healthy animals.22 In another, the authors used CRISPR to genetically edit nonhuman primate embryos, and subsequently found the presence of the targeted genetic mutation in the postnatal animal.23 Both of these studies found healthy offspring from the edited embryos. These are just two examples, as there are many other published studies using genetically modified embryos for studying various outcomes. For many of the animal embryo studies, a goal has been to produce animal models for the investigation of molecular disease mechanisms and genetic disorders by creating embryonic genetic modifications and then studying the effects of these mutations in the live offspring.

Human embryonic gene editing studies are still in their infancy stage, but these types of studies will most certainly continue. Support for this observation are the number of ongoing discussions in the literature suggesting additional human embryonic gene editing studies are either in progress or may have been finished and are now awaiting review and publication.

Gene Editing and Potential Clinical Applications

Numerous studies from the last century have estimated there are nearly 10,000 single gene (monogenic) human diseases, and have also shown genetic risk variants in nearly every major complex human disease, including cardiovascular diseases, diabetes, and cancer.24 Our improved understanding of human genetics and therapeutics have led to the successful development of conventional therapies for a significant number of these disorders; however, for most genetic-based diseases we continue to lack “tools” for any type of successful therapeutic intervention. Gene therapy has long been promoted as the solution to this problem, but the field is only now beginning to bear fruit. A few early gene therapy trials reported some clinical benefits, but as stated earlier, a number of these patients experienced unacceptable adverse effects. More recent phase I and phase II trials targeting tissues in the retina, blood, and liver have been more successful due to improved vector design and delivery.25 This is an appropriate solution for repairing some of the genetic-based metabolic disorders but not all. For many, the only solution seems to be to correct the mutation at the gene level using site-directed endonucleases, such as ZFNs and CRISPR. Some of these studies and clinical trials have already been completed and others are in progress, including one in the United States using CRISPR technology.26

Some Thoughts about the Future of Gene Editing Technology

The seemingly exponential advances in genetic modification technology should enable the further deciphering of the many roles that the genome plays in our lives, as well as help elucidate how our genes might figure into the basic mechanisms of disease susceptibility and onset. These technologies should also assist the biotech/biopharma world in designing new drugs to treat disease not only at the organ/systemic level but also at the level of the gene itself. Major hurdles to its therapeutic use remain, as many questions must be answered before gene editing can become of use at the clinical level. Many of these questions relate to issues already discussed above, particularly those related to the safety and efficacy of the gene editing technology. Other questions not specific to the technology will include those related to how the genetically modified material will be handled by the “host” and its innate defense mechanisms once it enters the patient. This is probably of more concern if the genetically modified “foreign” material is from an unrelated donor (allogenic) versus from the same patient (autologous), but given the immune system’s dislike for anything foreign, both may become a target of the host defense systems. These are but a mere sampling of the many questions that must be answered, which may be solved quickly—at least in science terms—given the current rate of advances in gene editing technology and the rate at which clinical trials using these techniques are springing up worldwide.

Although somatic cell gene editing is quickly moving from the laboratory to the clinical trial stage, the more controversial aspect of human gene editing continues to be in its potential use for modifying disease states at the embryonic level. At least in this country, whether the use of embryonic gene modifications makes it to the clinical world will involve not only the many scientific hurdles that embryonic editing presents, but also ethics, political issues, and society norms that will need to be addressed (and solved) before embryonic editing will make its way into the laboratory, let alone clinical trials. Advances in human embryo gene editing will most likely come from scientists in countries outside the United States, where much of this type of research is currently being conducted.

What’s next in the gene editing story? Although the answer would be pure speculation, it is not a matter of if gene editing will become a part of clinical medicine but when. Another certainty is the legal profession will have a significant role to play in this ongoing story.


1. Dan M. Roden & Alfred L. George Jr., The Genetic Basis of Variability in Drug Responses, 1 Nature Revs. Drug Discovery 37 (2002).

2. FDA, Paving the Way for Personalized Medicine: FDA’s Role in a New Era of Medical Product Development (2013), available at Topics/PersonalizedMedicine/UCM372421.pdf.

3. R. Michael Blaese et al., T Lymphocyte-Directed Gene Therapy for ADA-SCID: Initial Trial Results after 4 Years, 270 Sci. 475 (1995).

4. Kirk R. Thomas & Mario R. Capecchi, Introduction of Homologous DNA Sequences into Mammalian Cells Induces Mutations in the Cognate Gene, 324 Nature 34 (1986).

5. Marina Bibikova et al., Enhancing Gene Targeting with Designed Zinc Finger Nucleases, 300 Sci. 764 (2003).

6. Bjoern Petersen & Heiner Niemann, Advances in Genetic Modification of Farm Animals Using Zinc-Finger Nucleases (ZFN), 23 Chromosome Res. 7 (2015); Joseph F. Petolino, Genome Editing in Plants via Designed Zinc Finger Nucleases, 51 In Vitro Cellular & Developmental Biology: Plant 1 (2015); Fyodor D. Urnov et al., Highly Efficient Endogenous Human Gene Correction Using Designed Zinc-Finger Nucleases, 435 Nature 646 (2005).

7. Michelle Christain et al., Targeting DNA Double-Strand Breaks with TAL Effector Nucleases, 186 Genetics 757 (2010).

8. Le Cong et al., Multiplex Genome Engineering Using CRISPR/Cas Systems, 339 Sci. 819 (2013); Martin Jinek et al., A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity, 337 Sci. 816 (2012); Prashant Mali et al., RNA-Guided Human Genome Engineering via Cas9, 339 Sci. 823 (2013).

9. Cong et al., supra note 8; Jinek et al., supra note 8; Mali et al., supra note 8; Haoyi Wang et al., One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering, 154 Cell 910 (2013).

10. Yanfang Fu et al., High-Frequency Off-Target Mutagenesis Induced by CRISPR-Cas Nucleases in Human Cells, 31 Nature Biotechnology 822 (2013); Takeshi Maruyama et al., Increasing the Efficiency of Precise Genome Editing with CRISPR-Cas9 by Inhibition of Nonhomologous End Joining, 33 Nature Biotechnology 538 (2015).

11. Ayal Hendel et al., Chemically Modified Guide RNAs Enhance CRISPR-Cas Genome Editing in Human Primary Cells, 33 Nature Biotechnology 985 (2015); Kathrin Schumann et al., Generation of Knock-In Primary Human T Cells Using Cas9 Ribonucleoproteins, 112 PNAS 10,437 (2015).

12. Alexander G. Bassuk et al., Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells, 6 Sci. Rep. 19,969 (2016); Jerry R. Mendell & Louise R. Rodino-Klapac, Duchenne Muscular Dystrophy: CRISPR/Cas9 Treatment, 26 Cell Res. 513 (2016).

13. Morgan L. Maeder & Charles A. Gersbach, Genome-Editing Technologies for Gene and Cell Therapy, 24 Molecular Therapy 430 (2016).

14. Id.

15. F. Ann Ran et al., Genome Engineering Using the CRISPR-Cas9 System, 8 Nature Protocols 2281 (2013).

16. Maeder & Gersbach, supra note 13.

17. Patrick T. Harrison et al., Impact of Gene Editing on the Study of Cystic Fibrosis, 135 Hum. Genetics 983 (2016); Ipek Tasan et al., Use of Genome-Editing Tools to Treat Sickle Cell Disease, 135 Hum. Genetics 1011 (2016).

18. Marie F. La Russa & Lei S. Qi, The New State of the Art: Cas9 for Gene Activation and Repression, 35 Molecular & Cellular Biology 3800 (2015).

19. Ewen Callaway, Gene-Editing Research in Human Embryos Gains Momentum, 532 Nature 289 (2016); Erik Stokstad, U.K. Researcher Details Proposal for CRISPR Editing of Human Embryos, Sci. (Jan. 13, 2016), editing-human-embryos.

20. Puping Liang et al., CRISPR/Cas9-Mediated Gene Editing in Human Tripronuclear Zygotes, 6 Protein & Cell 363 (2015).

21. Xiangjin Kang et al., Introducing Precise Genetic Modifications into Human 3PN Embryos by CRISPR/Cas-Mediated Genome Editing, 33 J. Assisted Reprod. & Genetics 581 (2016).

22. Chengzu Long et al., Prevention of Muscular Dystrophy in Mice by CRISPR/Cas9-Mediated Editing of Germline DNA, 345 Sci. 1184 (2014).

23. Yuyu Niu et al., Generation of Gene-Modified Cynomolgus Monkeys via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos, 156 Cell 836 (2014).

24. Norihiro Kato, Insights into the Genetic Basis of Type 2 Diabetes, 4 J. Diabetes Investigation 233 (2013); Christopher J. O’Donnell & Elizabeth G. Nabel, Genomics of Cardiovascular Disease, 365 New Eng. J. Med. 2098 (2011); Alkes L. Price et al., Progress and Promise in Understanding the Genetic Basis of Common Diseases, 282 Proc. Royal Soc’y B 20151684 (2015).

25. Luigi Naldini, Gene Therapy Returns to Centre Stage, 536 Nature 351 (2015).

26. Pablo Tebas et al., Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV, 370 New Eng. J. Med. 901 (2014); David Cyranoski, Chinese Scientists to Pioneer First Human CRISPR Trial, Nature (July 21, 2016),; Sara Reardon, First CRISPR Clinical Trial Gets Green Light from US Panel, Nature (June 22, 2016),; Karen Zusi, Gene Editing Treats Leukemia, Scientist (Nov. 6, 2015),