Current state of Gene Therapy

Current state of Gene Therapy

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I am interested in learning about attempts to treat adult individuals suffering from a genetic disease in which the underlying changes in the DNA of the gene are understood.

(i) Are there approaches that use drugs or do they use DNA corresponding to the normal DNA?

(ii) Are there attempts to reverse the condition or only ammeliorate it?

(iii) How near is such gene therapy to clinical use and for which diseases?

The scope of this question is too wide to be answered on Biology SE. However I will give you very brief answers to your questions (as I have rephrased them) and point you towards some sources of basic information on the Internet. After reading these you may wish to return with more specific questions. I have also briefly summarized some of the problems which it is useful to be aware of before reading details of specific approaches to gene therapy,.


(i) Some approaches involve attempts to replace a defective gene with a functional gene or add the latter to the patient's DNA. Others involve drugs, often nucleic acid in nature.

(ii) Current efforts often have the more modest aim of ameliorating the disease, although there are also efforts to reverse the condition.

(iii) Gene therapy is not yet in general clinical use, although for some diseases (e.g. Severe Combined Immune Deficiency and Chronic Granulomatus Disorder) there have been reports of success in limited clinical trials.

General Reading

I am not an expert in this area, but by Googling for 'Gene Therapy' I came up with the following pages that you may find helpful.

Your Genome

University of Utah: Approaches to Gene Therapy

American Society of Cell and Gene Therapy

You may also be interested to read about the currently controversial drug, Eteplirsen.

Problems in Gene Therapy

Rather than giving you details of technical approaches to gene therapy, for which you may not have the necessary scientific background, I draw your attention to some of the key problems and questions that have to be addressed in this area.

  1. Does one need to counteract the harmful effect of a mutant gene product (e.g. sickle cell haemoglobin), or is it sufficient to produce a 'good' gene product to remedy a deficit?
  2. Is it possible to target new cells or does one have to deal with existing mature cells? (Some cells like blood and immune cells are constantly renewing themselves, whereas others like muscle turn over much more slowly. The latter are more difficult to deal with.)
  3. How can the DNA (or drug) be deliverdd to the cell, and only to the appropriate cell. (A variety of delivery techniques have been employed in different circumstances.)
  4. How can the therapeutic DNA be incorporated into the appropriate position in the genome, and how can one ensure that it is expressed, and that no other genes are affected. (Early attempts at gene therapy using in which DNA was inserted at the wrong position resulted in cancers. However technology here is moving rapidly.)

Recent developments and current status of gene therapy using viral vectors in the United Kingdom

Viral vectors remain the vehicles of choice for gene transfer in clinical trials of gene therapy in the United Kingdom and worldwide. In the United Kingdom alone, 88 clinical trials are registered with the Gene Therapy Advisory Committee (GTAC,

Although notable successes have been achieved in some inherited diseases, viral vectors have not been without their problems. We review the adverse events encountered to date and describe lessons that can be learnt from them to improve vector biology and pharmacology in order to make gene therapy with viral vectors a viable treatment for the future.

A new gene therapy strategy, courtesy of nature

Scientists have developed a new gene-therapy technique by transforming human cells into mass producers of tiny nano-sized particles full of genetic material that has the potential to reverse disease processes.

Though the research was intended as a proof of concept, the experimental therapy slowed tumor growth and prolonged survival in mice with gliomas, which constitute about 80 percent of malignant brain tumors in humans.

The technique takes advantage of exosomes, fluid-filled sacs that cells release as a way to communicate with other cells.

While exosomes are gaining ground as biologically friendly carriers of therapeutic materials -- because there are a lot of them and they don't prompt an immune response -- the trick with gene therapy is finding a way to fit those comparatively large genetic instructions inside their tiny bodies on a scale that will have a therapeutic effect.

This new method relies on patented technology that prompts donated human cells such as adult stem cells to spit out millions of exosomes that, after being collected and purified, function as nanocarriers containing a drug. When they are injected into the bloodstream, they know exactly where in the body to find their target -- even if it's in the brain.

"Think of them like Christmas gifts: The gift is inside a wrapped container that is postage paid and ready to go," said senior study author L. James Lee, professor emeritus of chemical and biomolecular engineering at The Ohio State University.

And they are gifts that keep on giving, Lee noted: "This is a Mother Nature-induced therapeutic nanoparticle."

The study is published today (Dec. 16) in the journal Nature Biomedical Engineering.

In 2017, Lee and colleagues made waves with news of a regenerative medicine discovery called tissue nanotransfection (TNT). The technique uses a nanotechnology-based chip to deliver biological cargo directly into skin, an action that converts adult cells into any cell type of interest for treatment within a patient's own body.

By looking further into the mechanism behind TNT's success, scientists in Lee's lab discovered that exosomes were the secret to delivering regenerative goods to tissue far below the skin's surface.

The technology was adapted in this study into a technique first author Zhaogang Yang, a former Ohio State postdoctoral researcher now at the University of Texas Southwestern Medical Center, termed cellular nanoporation.

The scientists placed about 1 million donated cells (such as mesenchymal cells collected from human fat) on a nano-engineered silicon wafer and used an electrical stimulus to inject synthetic DNA into the donor cells. As a result of this DNA force-feeding, as Lee described it, the cells need to eject unwanted material as part of DNA transcribed messenger RNA and repair holes that have been poked in their membranes.

"They kill two birds with one stone: They fix the leakage to the cell membrane and dump the garbage out," Lee said. "The garbage bag they throw out is the exosome. What's expelled from the cell is our drug."

The electrical stimulation had a bonus effect of a thousand-fold increase of therapeutic genes in a large number of exosomes released by the cells, a sign that the technology is scalable to produce enough nanoparticles for use in humans.

Essential to any gene therapy, of course, is knowing what genes need to be delivered to fix a medical problem. For this work, the researchers chose to test the results on glioma brain tumors by delivering a gene called PTEN, a cancer-suppressor gene. Mutations of PTEN that turn off that suppression role can allow cancer cells to grow unchecked.

For Lee, founder of Ohio State's Center for Affordable Nanoengineering of Polymeric Biomedical Devices, producing the gene is the easy part. The synthetic DNA force-fed to donor cells is copied into a new molecule consisting of messenger RNA, which contains the instructions needed to produce a specific protein. Each exosome bubble containing messenger RNA is transformed into a nanoparticle ready for transport, with no blood-brain barrier to worry about.

"The advantage of this is there is no toxicity, nothing to provoke an immune response," said Lee, also a member of Ohio State's Comprehensive Cancer Center. "Exosomes go almost everywhere in the body, including passing the blood-brain barrier. Most drugs can't go to the brain.

"We don't want the exosomes to go to the wrong place. They're programmed not only to kill cancer cells, but to know where to go to find the cancer cells. You don't want to kill the good guys."

The testing in mice showed the labeled exosomes were far more likely to travel to the brain tumors and slow their growth compared to substances used as controls.

Because of exosomes' safe access to the brain, Lee said, this drug-delivery system has promise for future applications in neurological diseases such as Alzheimer's and Parkinson's disease.

"Hopefully, one day this can be used for medical needs," Lee said. "We've provided the method. If somebody knows what kind of gene combination can cure a certain disease but they need a therapy, here it is."

This work was supported by the National Science Foundation the National Natural Science Foundation of China the National Heart, Lung, and Blood Institute the National Institute of Neurological Disorders and Stroke the Cancer Prevention and Research Institute of Texas, the American Brain Tumor Association and the National Cancer Institute.

Ohio State co-authors Junfeng Shi, Jingyao Sun, Xinmei Wang, Yifan Ma, Veysi Malkoc, Chiling Chiang, Kwang Kwak, Yamin Fan, Paul Bertani, Jose Otero and Wu Lu also worked on the research.

Attempts to Improve Vector Design

Modifications of rAd vectors have been made in an attempt to decrease immunogenicity and increase cell-specificity and efficiency of gene transfer. The first-generation vectors used in the rAd-OTC, rAd-p53, and similar studies were deleted only for portions of their E1a and (for some) E3 coding regions. These vectors contained all of the other viral genes. Although E1a functions as a master-switch for activation of other genes, these vectors were “leaky” for expression of other early and late genes, which may have increased their immogenicity somewhat. Second generation vectors were designed that were deleted for E2a and E4 regions, and the latest helper-dependent (HD) Ad vectors are completely gutted, that is, possess no viral coding sequences (Yang et al., 1994 Engelhardt et al., 1994a , b Gao et al., 1996 Raper et al., 1998 Alba et al., 2005 Schiedner et al., 1998 ). While these vectors have elicited much less adaptive cytotoxic T-cell responses, it remains to be seen whether there will be much effect on innate cytokine responses. More recently, there has been considerable effort made to modify the Ad capsid, either by utilizing other Ad serotypes or by making targeted mutations with insertion of specific ligands to target particular cell-surface receptors (Wickham et al., 1997 Krasnykh et al., 1998 Dmitriev et al., 1998 Romanczuk et al., 1999 ). Increasing the cell-specificity of Ad attachment could have a major effect on innate immunity and increase the efficiency of gene transfer, thus decreasing the dose that would be needed for the desired effect. Once again, the clinical safety and efficacy of such modified vectors remains unproven.

Much as with rAd vectors, rAAV vectors have been subjected to targeted mutation. Receptor-specific ligands have been inserted into specific regions of the capsid gene so that they are then displayed on the surface of the capsid (Wu et al., 2000 Nicklin and Baker, 2002 Loiler et al., 2003 , 2005 Nicklin et al., 2003 White et al., 2004 Work et al., 2004 ). Work is also progressing with rAAV vectors that are based on newly available serotypes and genomic variants of AAV (Chao et al., 2000 Gao et al., 2002 Rabinowitz et al., 2002 ). The first 10 years of clinical rAAV gene therapy relied on serotype 2 vectors. These vectors benefited from the natural biology of AAV, a non-pathogenic human parvovirus that can infect non-dividing cells and remain latent for prolonged periods, predominantly in an episomal state (Afione et al., 1996 Flotte and Berns, 2005 ). AAV appears to persist in cells without causing insertional mutagenesis and does not trigger a robust innate response. It has a very limited packaging capacity, however (approximately 5 kb), and the original AAV2 is relatively inefficient at infecting a number of key cell types. Studies are now underway in the US and Europe with rAAV1 vectors, which have been shown to be nearly 1,000-fold more potent for murine muscle transduction than rAAV2 vector (Chao et al., 2000 Lu et al., 2006 ). Over 100 capsid variants are now known to exist (Gao et al., 2002 , 2003 ). These vary widely in tissue tropism and may have differences in their ability to trigger innate and adaptive immune responses.

Finally, rAAV has been subjected to in vitro evolution, using DNA shuffling between available serotype sequences to create a library of novel genomic variants (Schaffer and Maheshri, 2004 Koerber et al., 2006 Maheshri et al., 2006 Perabo et al., 2006 ). With the advent of robust methods to examine the crystal structure of such mutants, further in vitro evolution and serotype selection has been targeted to sites that are known to be displayed on the surface and with known effects on vector stability or infectivity (Fig. 1 Wu et al., 2006 ). Serial passage of such mutant libraries on a target cell type of interest, or even a target tissue in a mouse model, has allowed for selection of mutants with increased specificity and efficiency and decreased susceptibility to neutralizing antibodies.

Mapping of critical capsid mutant residues to the AAV2-VP3 crystal structure. (Reproduced with permission from Wu, et al., J. Virology 2006 80:11393–11397.) The three-dimensional surface maps and charge distribution of a VP3 trimer (one face of the icosahedral capsid) are compared between AAV2, AAV6, and AAV1 (parts a, b, and c, respectively).

Work is also progressing with clinical applications of lentivirus, herpesvirus, and newer non-viral gene therapy vectors. Lentivirus vectors offer similar advantages to the gamma-retrovirus vectors, in that they mediate long-term integration of the therapeutic transgene, but unlike gamma-retroviruses, they do not require cellular mitosis in order to gain access to the host genome for integration. They also are thought to share the potential for insertional mutagenesis with subsequent carcinogenesis, although this has not been observed in the limited number of clinical trials done to this point. They have also been successfully targeted by pseudotyping alternative surface glycoproteins for display on the lipid envelope of the vector.

Herpes simplex virus (HSV)-based vectors are large DNA-virus vectors that may trigger innate and adaptive immunity like rAd vectors, but they have a longer-term expression profile and can also infect non-dividing cells. These vectors have also gone through a series of iterations, including HSV amplicon vectors, which are HD and HSV recombinant vectors, which are conditionally replication deficient, but still may express low levels of the HSV proteins.

The non-viral systems that have been used for gene transfer are numerous (Niidome and Huang, 2002 ). Naked DNA alone is capable of significant gene transfer to skeletal muscle when directly injected (Wolff et al., 1992 ), and in mice can also transduce the liver when injected rapidly under high pressure into the subdiaphragmatic venous system (Knapp and Liu, 2004 Zhang et al., 2004 ). Naked plasmid DNA has been used for recombinant vaccine studies and for direct injection of a variety of genes into tumors. Numerous cationic liposomes have been used in clinical gene transfer, as well, either alone or as mixtures with neutral “co-lipids.” DNA conjugates with other poly-cationic molecules, such as polyamines, poly-lysine have also been used in clinical studies, either as a compacted DNA alone or in a complex with targeting peptides (Konstan et al., 2004 ). The latter modifications are in many ways analogous to the capsid targeting strategy described above for viral vectors. In addition, the safety of non-viral vector have been improved by the remove of CpG motifs which were the primary cause of innate responses through their interaction with toll-like receptors on the cell surface (Tan et al., 1999 Liu et al., 2004 ).

Gene Therapy: Principles and Applications | Genetics

In this article we will discuss about the principles and applications of gene therapy based on targeted inhibition of gene expression in vivo.

Principles and Applications of Therapy Based on Targeted Inhibition of Gene Expression In Vivo:

One way of treating certain human disor­ders is to selectively inhibit the expression of a predetermined gene in vivo. In principle, this general approach is particularly suited to treat­ing cancers and infectious diseases, and some immunological disorders.

In such cases, the basis of the therapy is to knock out the expres­sion of a specific gene that allows the cancer­ous cells, infection, allergy, inflammation, etc., to flourish, without interfering with normal cell function. For example, attention could be focused on selectively inhibiting the expression of a particular viral gene that is necessary for viral replication, or an inappropriately activa­ted oncogene, etc.

In addition to the above, targeted inhibi­tion of gene expression may offer the possibi­lity of treating certain dominantly inherited dis­orders. If a dominantly inherited disorder is the result of a loss-of function mutation, treatment may be possible using conventional gene augmentation therapy.

However, since hetero- zygotes with 50% of normal gene product can be severely affected, successful gene therapy for heterozygotes requires efficient expression of the introduced genes. However, dominantly inherited disorders which arise because of gain-of-function mutations may not be amenable to simple addition of normal genes.

Instead, it may be possible, in some cases, to specifically inhibit the expression of the mutant gene, but the expression of the normal allele must be maintained. Such allele-specific inhibition of gene expression is facilitated if the pathogenic mutation results in a significant sequence difference between the alleles.

The expression of a selected gene might be inhibited by a variety of different strategies. One possible type of approach involves spe­cific in vivo mutagenesis of that gene, altering it to a form that is no longer function. Gene targeting by homologous recombination offers the possibility of site-specific mutagenesis to inactivate a gene.

However, this technique has only very recently become feasible with nor­mal diploid somatic cells and is still very ineffi­cient. Instead, methods of blocking the expres­sion of a gene without mutating it are current­ly preferred.

In principle, this can be accom­plished at different levels: at the DNA level (by blocking transcription) at the RNA level (by blocking post-transcriptional processing, mRNA transport or engagement of the mRNA with the ribosomes) or at the protein level (by blocking post-translational processing, protein export or other steps that are crucial to the function of the protein).

Therapy by selective inhibition of gene expression is technically possible at all three expression levels (see Fig. 23.7):

(i) Triple helix therapeutics (involves binding of gene-specific oligonucleotides to double-stranded DNA in order to inhibit trans­cription of a gene).

(ii) Antisense therapeutics (involves binding of gene-specific oligonucleotides or polynucleotides to the RNA in some cases, the binding agent may be a specifically engi­neered ribozytne, a catalytic RNA molecule that can cleave the RNA transcript).

(iii) Use of intracellular antibodies (intra-bodies) and oligonucleotide aptamers (involves the construction of antibodies that can be directed to specific locations within cells in order to bind a specific protein, or oligonucleotide aptamers, which can bind specifically to a selected polypeptide).

Triple helix therapeutics relies on bind­ing of gene-specific oligonucleotides to the major groove of the double helix:

Synthetic short oligonucleotides (15-27 nucleotides long) are capable of specifically binding to a sequence of double-stranded DNA, forming a triple helix. The oligonu­cleotide binds by Hoogsteen hydrogen bonds to the double-stranded DNA, without disrupting the original Watson-Crick hydrogen bonding.

The most stable Hoogsteen-bonded structures are G bound to a GC base pair and a T bound to an AT base pair (see Fig. 23.8). Although such structures can inhibit DNA replication in vitro, helicases can unwind triple strand structures in vivo.

However, triplex for­mation has been shown to block binding of transcription factors in vitro, and also, at least in some cases, evidence has been obtained for gene-specific inhibition of transcription in intact cells.

Oligonucleotides are large polyanionic hydrophilic structures and so are not ideally suited to diffusing across the highly hydropho­bic plasma membrane. Direct delivery into the cytoplasm using cell permeabilization tech­niques provides the most efficient approach to enable subsequent transfer into the nucleus, and delivery using liposomes is a popularly used method.

Thereafter, the oligonucleotides can migrate rapidly to the nucleus (by passive diffusion through the pores of the nuclear envelope). Inside the cell, the oligonucleotides are exposed to nuclease attack, notably from exonucleases, and the half-life of conventional oligonucleotides with phosphodiester bonds is typically about 20 min.

Accordingly, it is usual for the 3′ and 5′ ends of the oligonucleotides to be chemically modified to protect against nuclease attack. Often, chemical modification involves incorporation of sulfur-containing phosphorothioate bonds to generate so- called S-oligonucleotides.

Although the technology is improving rapidly, some general difficulties need to be overcome. Inhibition of gene expression requires comparatively large amounts of oligonucleotide. More worrying is the limita­tion imposed by Hoogsteen hydrogen bond­ing the target sequences need to carry virtual­ly all their purine bases on one DNA strand. Preliminary attempts to solve this problem include replacement of the phosphate groups by different chemical groupings that allow triplex-forming oligonucleotides to ‘hop’ from one strand of the bound DNA duplex to the other.

Antisense oligonucleotides or poly­nucleotides can bind to a specific mRNA, inhibiting its translation and, in some cases, ensuring its destruction:

During transcription, only one of the two DNA strands in a DNA duplex, the template strand, serves as a template for making a complementary RNA molecule. As a result, the base sequence of the single-stranded RNA transcript is essentially identical (except that U replaces T) to the other DNA strand, com­monly called the sense strand.

Any oligonu­cleotide or polynucleotide which is comple­mentary in sequence to an mRNA sequence, including the templates strand of the gene, can, therefore, be considered to be an anti- sense sequence.

Binding of an antisense sequence to the corresponding mRNA sequence would be expected to interfere with translation, and thereby inhibit polypeptide synthesis. Indeed, naturally occurring antisense RNA is known to provide a way of regulating the expression of genes in some plant and animal cells, as well as in some microbes. Synthetic oligonu­cleotides can be designed to be complemen­tary in sequence to a specific mRNA and, when transferred into cells, show evidence of inhibition of expression of the corresponding gene.

As a result, the concept of antisense therapeutics was developed unwanted expression of a specific gene in disease tissues could be selectively inhibited using an artificia­lly gene-specific antisense sequence. A variety of different types of antisense sequence can be used.

Antisense Oligodeoxynucleotides:

The use of artificial antisense oligonu­cleotides is often favored, simply because they can be synthesized so simply. They can be transferred efficiently into the cytoplasm of cells using liposomes, and their intracellular stability is improved by using chemically modi­fied oligonucleotides, notably S-oligonucleo­tides (see above note that although antisense oligonucleotides migrate to the nucleus, they do not bind the double-stranded DNA because they are not designed to participate in Hoogsteem hydrogen bonding).

Antisense oligodeoxynucleotides (ODNs) are preferred to oligoribonucleotides because they are general­ly less vulnerable to nuclease attack, and importantly because they have the additional advantage of inducing the destruction of an mRNA to which they bind.

This is so because an ODN-mRNA hybrid, like all DNA-RNA hybrids, is vulnerable to attack and selective cleavage of the RNA strand by a specific class of intracellular ribonuclease, RNase H. Despite teething problems in early studies, refinement of the technology has meant that antisense ODNs are now considered to have great therapeutic potential, and clinical trials are now in progress for several human diseases

Antisense Genes:

Antisense oligonucleotides, even when chemically modified, are not stable indefini­tely. One way of ensuring a continuous supply of antisense sequence is a form of expression cloning in which a specially designed antisense gene is transferred into the relevant cells. Such a gene can be engineered simply by constructing a mini- gene in which an inverted coding sequence is placed downstream of a powerful promoter.

The DNA strand that normally serves as the sense strand is now transcribed to give an antisense RNA which can be synthesized repeatedly (Fig. 23.9). If the antisense gene is provided using an integrative vector, long- term production of antisense RNA may be obtained.


Increasingly, it is becoming clear that RNA molecules are functionally different from DNA molecules collectively they can serve diverse functions, rather than simply being involved in transfer of genetic informa­tion. Some RNA molecules are able to lower the activation energy for specific biochemical reactions, and so effectively function as enzymes (ribozymes). For example, the tran­scripts of group 1 introns are autocatalytic and self-splicing.

Other ribozymes which cleave RNA, are trans-acting, that is they cleave an RNA sequence on a different molecule. They contain two essential compo­nents target recognition sequences (which base-pair with complementary sequences on target RNA molecules), and a catalytic com­ponent, much like the active site of an enzyme (which cleaves the target RNA molecule while the base-pairing holds it in place).

The cleavage leads to inactivation of the RNA, presumably because of subsequent recognition by intracellular nucleases of the two unnatural ends. Examples include human ribonuclease P and various ribozymes obtained from plant viroids (virus-like parti­cles).

Genetic engineering can be employed to custom design the recognition sequence so that it contains antisense sequences that can base-pair to a specific mRNA molecule, while retaining the catalytic site (see Fig. 23.10). Engineered genes which can be transcribed to produce the desired ribozyme can then be transfected into suitable cells.

One applica­tion has been the design of ribozymes against specific oncoproteins. Pilot studies have shown that transfection of an anti-Ras ribozyme gene into human bladder carcino­ma cells with the ras mutation resulted in blocking of Ras production and reversal of the metastatic, invasive and tumorigenic properties of the cells. Early problems in the efficiency of targeting to their targets inside the cell are currently being addressed and clinical trials have already been initiated in some cases, such as in gene therapy for AIDS.

Artificially designed intracellular antibo­dies (intra-bodies), oligonucleotides (aptamers) and mutant proteins can inhibit the function of a specific polypeptide:

Intracellular Antibodies (Intra-bodies):

Antibody function is normally conducted extracellular: upon synthesis, antibodies are normally secreted into the extracellular fluid or remain membrane bound on the B-cell surface as antigen receptors. Recently, however, anti­body engineering has been extended to the design of genes encoding intracellular antibod­ies, or intra-bodies.

This achievement raises the possibility of using antibodies within cells to block the construction of viruses or harmful proteins, such as oncoproteins. The first exam­ple of this approach involved engineering the antibody F105 which binds to gp120, a crucial human immunodeficiency virus (HIV) envelope protein that the AIDS virus uses to attach to and infect its target cells.

This envelope protein is derived from a larger precursor gp160 which is synthesized in the endoplasmic reticulum. Marasco and co-work­ers designed a novel F105 gene which encod­ed an antibody that was stably expressed and retained in the endoplasmic reticulum without being toxic to the cells. The engineered anti­body binds to the HIV envelope protein within the cell and inhibits processing of the gpl60 precursor, thereby substantially reducing the infectivity of the HIV-1 particles produced by the cell.

Oligonucleotide Aptamers:

Fully degenerate oligonucleotides can be synthesized by delivering 25% each of the four bases A, C, G and T at each base position dur­ing oligonucleotide synthesis. As a result, the number of sequence permutations which can be generated (4″ where n is the chosen length of oligonucleotide) can be enormous.

The result­ing mixture of oligonucleotides can be used to screen for the ability to bind to a selected target protein (protein epitope targeting). In prac­tice, the use of partial degenerate oligonu­cleotides is preferred so that the concentration of individual oligonucleotides is not too low.

In effect, this means simultaneous screening of many thousands of oligonucleotides, and so the chance of at least one epitope of the target pro­tein being specifically bound by an oligonu­cleotide can be high. The bound oligonucleotide (sometimes known as an adaptamer or aptamer) can be eluted from the protein and sequenced to identify the specific recognition sequence. Transfer of large amounts of a che­mically stabilized aptamer into cells can result in specific binding to a predetermined polypep­tide, thereby blocking its function.

An initial success was the identification of oligonucleotides that could bind to and inhibit the protease thrombin, which is part of the blood coagulation cascade .Thrombin functions in serum and extracellular applications of this type are no different, in principle, from standard drug therapy. However, the future use of oligonucleotide aptamers to inhibit specific intracellular pro­tein targets will inevitably involve genetic modification of cells, and can, therefore, be considered as a form of gene therapy.

Mutant Proteins:

Naturally occurring gain-of-function muta­tions can involve the production of a mutant polypeptide that binds to the wild-type pro­tein, inhibiting its function. In many such cases, the wild-type polypeptides naturally associate to form multimers, and incorpora­tion of a mutant protein inhibits this process.

In some cases, gene therapy may be possible by designing genes to encode a mutant protein that can specifically bind to and inhibit a pre­determined protein, such as a protein essential for the life-cycle of a pathogen. For example, one form of gene therapy for AIDS involves artificial production of a mutant HIV-1 protein in an attempt to inhibit multimerization of the viral core proteins.

Artificial correction of a pathogenic mutation in vivo is possible, in princi­ple, but is very inefficient and not read­ily amenable to clinical applications:

Certain disorders are not easy targets for gene therapy. For example, dominantly inhe­rited disorders where a simple mutation results in a pathogenic gain of function cannot be treated by gene augmentation therapy, and targeted inhibition of gene expression may be difficult to achieve. Target inhibition is best suited to inhibiting novel or inappropriate gene expression in human cells, for example expression of viral genes, oncogenes, etc. Expression of a gain-of-function mutant allele may need to be inhibited while retaining expression of a very similar wild-type allele.

If the mutant allele carries a significant change in sequence at the site of the mutation, it may be possible to achieve selective inhibition but if the change is a simple mutation, say a single nucleotide substitution, other approaches may be needed. One possible approach is targe­ted mutation correction by inserting some reagents into cells in order to change the mutant sequence back to a form that is compatible with normal function.

In principle, there are several different ways in which a specific mutation can be cor­rected selectively in vivo, mostly at the DNA level. One way is to use gene targeting tech­niques based on homologous recombination. Because this approach offers the ability to make site-specific modifications of endoge­nous genes, it represents a potentially power­ful method for gene therapy: both acquired and inherited mutations could be corrected, and novel alterations could be engineered into the genome.

Thus far, this technique has been limited largely to pluripotent mouse embryon­ic stem cells, although recently it has been applied to normal diploid somatic cells .However, the enor­mous inefficiency of this procedure (even when using the ideal target of cells cultured in vitro) and the need to correct the defect in many different cells in vivo has meant that clinical applications are a long way off.

An alternative approach is to repair the genetic defect at the RNA level. One possibil­ity is to use a therapeutic ribozyme. One method envisages using a class of ribozymes known as group I introns, which are distin­guished by their ability to fold into a very spe­cific shape, capable of both cutting and splic­ing RNA.

If a transcript has, for example, a nonsense or a missense mutation, it may be possible to design specific ribozymes that can cut the RNA upstream of the muta­tion and then splice in a corrected transcript, a form of trans-splicing (see Fig. 23.11). Thus far, this technology is in its infancy, and cat­alytic efficiency needs to be improved. Another possibility is therapeutic RNA edit­ing.

This involves using a complementary RNA oligonucleotide to bind specifically to a mutant transcript at the sequence containing the pathogenic point mutation, and an RNA editing enzyme, such as double-stranded RNA adenosine deaminase, to direct the desired base modification . Again this technology is in its infancy and formidable technical difficulties need to be overcome before clinical applications can be envisaged.

[email protected]

The concept of gene therapy was envisioned soon after the emergence of restriction endonucleases and subcloning of mammalian genes in phage and plasmids. Over the ensuing decades, vectors were developed, including nonviral methods, integrating virus vectors (gammaretrovirus and lentivirus), and non-integrating virus vectors (adenovirus, adeno-associated virus, and herpes simplex virus vectors). Preclinical data demonstrated potential efficacy in a broad range of animal models of human diseases, but clinical efficacy in humans remained elusive in most cases, even after decades of experience in over 1000 trials. Adverse effects from gene therapy have been observed in some cases, often because of viral vectors retaining some of the pathogenic potential of the viruses upon which they are based. Later generation vectors have been developed in which the safety and/or the efficiency of gene transfer has been improved. Most recently this work has involved alterations of vector envelope or capsid proteins either by insertion of ligands to target specific receptors or by directed evolution. The disease targets for gene therapy are multiple, but the most promising data have come from monogenic disorders. As the number of potential targets for gene therapy continues to increase, and a substantial number of trials continue with both the standard and the later generation vector systems, it is hoped that a therapeutic niche for gene therapy will emerge in the coming decades.

The State of Gene Therapy

Gene therapy has made tremendous strides over the past decade or so. The first approved drugs are on the market, and literally hundreds of trials are underway offering unprecedented hope to patients with rare, debilitating genetic disorders. Nevertheless, safety issues continue to swirl around the field as adverse events are reported in trials involving both lentivirus and AAV vectors. Pricing and manufacturing pose additional hurdles that could significantly challenge the field.

In the May edition of GEN Live, we discuss the transformation of gene therapy from an academic endeavor to a billion-dollar biotech industry. We’ll discuss the latest research advances and hurdles, highlights from ASGCT, trends in delivery, and hear the panel’s vision for the future.

Webinar produced with support from:

Heather Gray-Edwards, PhD
Assistant Professor, Radiology
UMass Medical School James M. Wilson MD, PhD
Director, Gene Therapy Program
Perelman School of Medicine, Penn Artur Padzik, PhD
AAV Production Manager

How Does Gene Therapy Work?

The gene therapy can be carried out ex vivo or in vivo. In the ex vivo approach, the intended genes are transferred into the cells grown in culture. Transformed cells are selected and then re-introduced into the patient. The in vivo approach involves the transfer of cloned genes directly into the tissues of the patient.

The process of gene therapy starts with the selection of a suitable vector, a carrier that will transfer the intended gene to the cells. Two types of vectors used in gene therapy are viral vectors—recombinant viruses, and non-viral vectors—naked DNA or DNA complexes. The viral vectors introduce their genetic material into the host cell and use the host cell’s machinery to produce proteins encoded by the viral DNA. Viruses used in gene therapy are retroviruses, adenoviruses, adeno-associated virus, herpes simplex, and vaccinia. Retroviruses are most commonly used because they can incorporate their genetic material into the host cell’s DNA, thus changing the genetic component of that cell. And they have an extremely high transfection frequency, enabling a large proportion of the stem cells in a bone marrow extract to receive the new gene.

As the vector binds and enters inside the target cell, its genetic material enters the cell’s nucleus. Then, the viral vector either tricks the host cell’s machinery to replicate its genetic material or integrate its genetic material into the host genome and cause it to replicate and produce proteins encoded by the viral genetic material. Thus, the therapeutic gene previously recombined with the viral genetic material can be expressed in the host cell.

Gene therapy techniques are applied with various strategies based on the need for function. Gene augmentation therapy is used to add a functioning gene into a cell with a non-functioning copy of that gene. It is suitable to treat diseases caused by a mutation that stops a gene from producing a functioning product, such as a protein. Similarly, Gene inhibition therapy is suitable for the treatment of cancer, infectious, and inherited diseases caused by improper gene activity. This does so by blocking the expression of a gene or interfering with the activity of the product of another gene. The third strategy is targeted killing of specific cells such as cancers by inserting suicide genes or genes encoding antigenic proteins. The fourth strategy is correcting a defective or mutant gene to restore its function.

Scientists Develop New Gene Therapy Strategy to Delay Aging and Extend Lifespan

Cellular senescence, a state of permanent growth arrest, has emerged as a hallmark and fundamental driver of organismal aging. It is regulated by both genetic and epigenetic factors. Despite a few previously reported aging-associated genes, the identity and roles of additional genes involved in the regulation of human cellular aging remain to be elucidated. Yet, there is a lack of systematic investigation on the intervention of these genes to treat aging and aging-related diseases.

How many aging-promoting genes are there in the human genome? What are the molecular mechanisms by which these genes regulate aging? Can gene therapy alleviate individual aging? Recently, researchers from the Chinese Academy of Sciences have shed new light on the regulation of aging.

Recently, researchers from the Institute of Zoology of the Chinese Academy of Sciences (CAS), Peking University, and Beijing Institute of Genomics of CAS have collaborated to identify new human senescence-promoting genes by using a genome-wide CRISPR/Cas9 screening system and provide a new therapeutic approach for treating aging and aging-related pathologies.

In this study, the researchers conducted genome-wide CRISPR/Cas9-based screens in human premature aging stem cells and identified more than 100 candidate senescence-promoting genes. They further verified the effectiveness of inactivating each of the top 50 candidate genes in promoting cellular rejuvenation using targeted sgRNAs.

Among them, KAT7 encoding a histone acetyltransferase was identified as one of the top targets in alleviating cellular senescence. It increased in human mesenchymal precursor cells during physiological and pathological aging. KAT7 depletion attenuated cellular senescence whereas KAT7 overexpression accelerated cellular senescence.

Mechanistically, inactivation of KAT7 decreased histone H3 lysine 14 acetylation, repressed p15 INK4b transcription, and rejuvenated senescent human stem cells.

Cumulative studies have described that age-associated accumulation of senescent cells and proinflammatory cells in tissues and organs contribute to the development and progression of aging as well as aging-related disorders. Prophylactic ablation of senescent cells mitigates tissue degeneration and extends the healthspan in mice.

In this study, the researchers found that intravenous injection of a lentiviral vector encoding Cas9/sg-KAT7 reduced the proportions of senescent cells and proinflammatory cells in the liver, diminished circulatory senescence-associated secretory phenotype (SASP) factors in the serum, and extended healthspan and lifespan of aged mice.

Figure. Gene therapy targeting Kat7 extends lifespan in naturally aged and progeria mice. Credit: IOZ

These results suggest that gene therapy based on single-factor inactivation may be sufficient to extend mouse lifespan. The researchers also found that the treatment with the lentiviral vector encoding Cas9/sg-KAT7 or a KAT7 inhibitor WM-3835 alleviated human hepatocyte senescence and reduced the expression of SASP genes, suggesting the possibility of applying these interventions in clinical settings.

Altogether, this study has successfully expanded the list of human senescence-promoting genes using CRISPR/Cas9 genome-wide screen and conceptually demonstrated that gene therapy based on single-factor inactivation is able to delay individual aging. This study not only deepens our understanding of aging mechanism but also provides new potential targets for aging interventions.

Types of Gene Therapy

The two gene therapy types are germ line gene therapy and somatic gene therapy. While the germ line type is aimed at permanent manipulation of genes in the germ cells, the somatic gene therapy refers to correction of genes in the somatic or body cells.

The two gene therapy types are germ line gene therapy and somatic gene therapy. While the germ line type is aimed at permanent manipulation of genes in the germ cells, the somatic gene therapy refers to correction of genes in the somatic or body cells.

The advancement of medical science has made it possible to treat some of the irreversible, life-threatening diseases, both in humans and animals. It is amazing to know that defective DNA (deoxyribonucleic acid) of a patient suffering from genetic diseases can be replaced or repaired to treat underlying medical problems. Such an approach is called gene therapy. Based on which cell is involved in the procedure, there are two different types of gene therapy.

Gene Therapy Basics

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In a cell, it is the stretch of DNA or gene that directs synthesis of specific proteins for cell expression and functioning. The sequence of nucleotides in the gene determines which type of protein is to be produced in what quantity. If there is an aberration in the sequence, the consequences are synthesis of unusual proteins, or no protein production. These in turn result in genetic disorders. Examples of the same are sickle-cell anemia, hemophilia, cystic fibrosis, Alzheimer’s disease and Down’s syndrome amongst others.

Gene therapy is defined as the aspect of therapeutic intervention, which involves the introduction of specific genes in a patient’s cell with the help of vectors (specially viruses) or repairing the faulty genes. Thus, the abnormal genes responsible for causing ailments are rectified by inserting new genes, repairing the defective genes or removing problematic genes, so as to restore normal functioning of the bodily systems. To be more precise, treatment is proceeded to the genetic level of the individual with the help of gene therapy.

Gene Therapy Types

The main objective of gene therapy is to correct genetic diseases and disorders, which are non-responsive to the regular treatment approaches. Say for instance, chemotherapy and radiation therapy are proceeded to kill cancerous cells, but not to combat the root cause (uncontrolled cell multiplication). Gene therapy for cancer treatment is directed at correcting the faulty genes (oncogenes) or blocking them to change to normal cells. Gene therapy is broadly classified into somatic and germ line gene therapy. These various types of gene therapy are explained below.

Germ Line Gene Therapy

In this gene therapy, the procedure is centered around permanent rectification of gene in the germ cells, i.e., the eggs or the sperms. Since these germ cells are passed on from the parents to their offspring, the corrected gene is inherited to the successive generations. If this gene therapy turns out to be successful, it will help in preventing transmission of genetic problems to the next generation, especially those which run in families.

Somatic Gene Therapy

Over here, the alteration of gene is conducted to the somatic or body cells (not the germ line cells). Since the targeted genes are not transmissible from one generation to another, it manipulates body cells to combat individual health condition. As expected, the gene alteration is temporary (just for the patient) and is not inherited further to the next generation. It is a conservative therapy, and side effects (if any) are restricted to the patient only.

In the two types of gene therapy, germ line type is relatively new, and more research is underway. Till date, it has been experimented in animal studies and not in humans. For both cases of gene therapy, the protocol steps remain nearly the same. The procedure encompasses any of the techniques – insertion of a normal gene, recombination for exchanging abnormal gene with a normal one, reverse mutation technique for reversion of faulty gene to normal one and altering regulatory processes of genes to control protein synthesis. Learn more on gene therapy pros and cons.

When it comes to the application of gene therapy procedure, it does hold a specific place in medical science. But, the downside story is, this gene manipulation is not free of drawbacks. Truly speaking, it is quite early to announce the promising scopes of gene therapy. Yes, both types of therapy are accompanied with some side effects, and further studies are needed to confirm satisfactory results.

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