Therapeutic gene modulation

From Wikipedia, the free encyclopedia

 

Therapeutic gene modulation refers to the practice of altering the expression of a gene at one of various stages, with a view to alleviate some form of ailment. It differs from gene therapy in that gene modulation seeks to alter the expression of an endogenous gene (perhaps through the introduction of a gene encoding a novel modulatory protein) whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly.

Modulation of gene expression can be mediated at the level of transcription by DNA-binding agents (which may be artificial transcription factors), small molecules, or synthetic oligonucleotides. It may also be mediated post-transcriptionally through RNA interference.

Transcriptional gene modulation

An approach to therapeutic modulation utilizes agents that modulate endogenous transcription by specifically targeting those genes at the gDNA level. The advantage to this approach over modulation at the mRNA or protein level is that every cell contains only a single gDNA copy. Thus the target copy number is significantly lower allowing the drugs to theoretically be administered at much lower doses.

This approach also offers several advantages over traditional gene therapy. Directly targeting endogenous transcription should yield correct relative expression of splice variants. In contrast, traditional gene therapy typically introduces a gene which can express only one transcript, rather than a set of stoichiometrically-expressed spliced transcript variants. Additionally, virally-introduced genes can be targeted for gene silencing by methylation which can counteract the effect of traditional gene therapy.^[1] This is not anticipated to be a problem for transcriptional modulation as it acts on endogenous DNA.

There are three major categories of agents that act as transcriptional gene modulators: triplex-forming oligonucleotides (TFOs), synthetic polyamides (SPAs), and DNA binding proteins.^[2]

Triplex-forming oligonucleotides

What are they

Triplex-forming oligonucleotides (TFO) are one potential method to achieve therapeutic gene modulation. TFOs are approximately 10-40 base pairs long and can bind in the major groove in duplex DNA which creates a third strand or a triple helix.^[2][3] The binding occurs at polypurine or polypyrimidine regions via Hoogsteen hydrogen bonds to the purine (A / G) bases on the double stranded DNA that is already in the form of the Watson-Crick helix.^[4]

How they work

TFOs can be either polypurine or polypyrimidine molecules and bind to one of the two strands in the double helix in either parallel or antiparallel orientation to target polypurine or polypyrimidine regions. Since the DNA-recognition codes are different for the parallel and the anti-parallel fashion of TFO binding, TFOs composed of pyrimidines (C / T) bind to the purine-rich strand of the target double helix via Hoogsteen hydrogen bonds in a parallel fashion.^[3] TFOs composed of purines (A / G), or mixed purine and pyrimidine bind to the same purine-rich strand via reverse Hoogsteen bonds in an anti-parallel fashion. TFO's can recognize purine-rich target strands for duplex DNA.^[2]

Triple Stranded DNA:TFOs bind in a similar fashion to the double stranded DNA as a triplex helix configuration.

Complications and limitations

In order for TFO motifs to bind in a parallel fashion and create hydrogen bonds, the nitrogen atom at position 3 on the cytosine residue needs to be protonated, but at physiological pH levels it is not, which could prevent parallel binding.^[2]

Another limitation is that TFOs can only bind to purine-rich target strands and this would limit the choice of endogenous gene target sites to polypurine-polypyrimidine stretches in duplex DNA. If a method to also allow TFOs to bind to pyrimidine bases was generated, this would enable TFOs to target any part of the genome. Also the human genome is rich in polypurine and polypyrimidine sequences which could affect the specificity of TFO to bind to a target DNA region. An approach to overcome this limitation is to develop TFOs with modified nucleotides that act as locked nucleic acids to increase the affinity of the TFO for specific target sequences.^[5]

Other limitations include concerns regarding binding affinity and specificity, in vivo stability, and uptake into cells. Researchers are attempting to overcome these limitations by improving TFO characteristics through chemical modifications, such as modifying the TFO backbone to reduce electrostatic repulsions between the TFO and the DNA duplex. Also due to their high molecular weight, uptake into cells is limited and some strategies to overcome this include DNA condensing agents, coupling of the TFO to hydrophobic residues like cholesterol, or cell permeabilization agents.^[2]

What can they do

Scientists are still refining the technology to turn TFOs into a therapeutic product and much of this revolves around their potential applications in antigene therapy. In particular they have been used as inducers of site-specific mutations, reagents that selectively and specifically cleave target DNA, and as modulators of gene expression.^[6] One such gene sequence modification method is through the targeting DNA with TFOs to active a target gene. If a target sequence is located between two inactive copies of a gene, DNA ligands, such as TFOs, can bind to the target site and would be recognized as DNA lesions. To fix these lesions, DNA repair complexes are assembled on the targeted sequence, the DNA is repaired. Damage of the intramolecular recombination substrate can then be repaired and detected if resection goes far enough to produce compatible ends on both sides of the cleavage site and then 3' overhangs are ligated leading to the formation of a single active copy of the gene and the loss of all the sequences between the two copies of the gene.^[4]

In model systems TFOs can inhibit gene expression at the DNA level as well as induce targeted mutagenesis in the model.^[6] TFO-induced inhibition of transcription elongation on endogenous targets have been tested on cell cultures with success.^[7] However, despite much in vitro success, there has been limited achievement in cellular applications potentially due to target accessibility.

TFOs have the potential to silence silence gene by targeting transcription initiation or elongation, arresting at the triplex binding sites, or introducing permanent changes in a target sequence via stimulating a cell's inherent repair pathways. These applications can be relevant in creating cancer therapies that inhibit gene expression at the DNA level. Since aberrant gene expression is a hallmark of cancer, modulating these endogenous genes' expression levels could potentially act as a therapy for multiple cancer types.

Synthetic polyamides

Synthetic polyamides are a set of small molecules that form specific hydrogen bonds to the minor groove of DNA. They can exert an effect either directly, by binding a regulatory region or transcribed region of a gene to modify transcription, or indirectly, by designed conjugation with another agent that makes alterations around the DNA target site.

Cartoon representation of a synthetic polyamide to DNA sequence recognition. The DNA sequence 5'-GTAC-3' is recognized by the amino acid pairs Py/Im, Py/Hp, Hp/Py, and Im/Py. See^[8][9] for chemical structure

Structure

Specific bases in the minor groove of DNA can be recognized and bound by small synthetic polyamides (SPAs). DNA-binding SPAs have been engineered to contain three polyamide amino acid components: hydroxypyrrole (Hp), imidazole (Im), and pyrrole (Py).^[10] Chains of these amino acids loop back on themselves in a hairpin structure. The amino acids on either side of the hairpin form a pair which can specifically recognize both sides of a Watson-Crick base pair. This occurs through hydrogen bonding within the minor groove of DNA. The amide pairs Py/Im, Py/Hp, Hp/Py, and Im/Py recognize the Watson-Crick base pairs C-G, A-T, T-A, and G-C, respectively (Table 1). See figure for a graphical representation of 5'-GTAC-3' recognition by a SPA. SPAs have low toxicity, but have not yet been used in human gene modulation.

Table 1. Amide pair to nucleotide pair recognition code.

Amide Pair	Nucleotide Pair
Py/Im	C-G
Py/Hp	A-T
Hp/Py	T-A
Im/Py	G-C

Limitations and workarounds

The major structural drawback to unmodified SPAs as gene modulators is that their recognition sequence cannot be extended beyond 5 Watson-Crick base pairings. The natural curvature of the DNA minor groove is too tight a turn for the hairpin structure to match. There are several groups with proposed workarounds to this problem.^{[8][11][12][13][14]} SPAs can be made to better follow the curvature of the minor groove by inserting beta-alanine which relaxes the structure.^[10] Another approach to extending the recognition length is to use several short hairpins in succession.^[15][16] This approach has increased the recognition length to up to eleven Watson-Crick base pairs.

Synthetic polyamide blocking of RNA transcription by binding within the transcribed region.

Synthetic polyamide blocking of transcription factors.

Direct modulation

SPAs may inhibit transcription through binding within a transcribed region of a target gene. This inhibition occurs through blocking of elongation by an RNA polymerase.

SPAs may also modulate transcription by targeting a transcription regulator binding site. If the regulator is an activator of transcription, this will decrease transcriptional levels. As an example, SPA targeting to the binding site for the activating transcription factor TFIIIA has been demonstrated to inhibit transcription of the downstream 5S RNA.^[17] In contrast, if the regulator is a repressor, this will increase transcriptional levels. As an example, SPA targeting to the host factor LSF, which represses expression of the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR), blocks binding of LSF and consequently de-represses expression of LTR^[18] .

Synthetic polyamide conjugated to modifying agent.

Conjugate modulation

SPAs have not been shown to directly modify DNA or have activity other than direct blocking of other factors or processes. However, modifying agents can be bound to the tail ends of the hairpin structure. The specific binding of the SPA to DNA allows for site-specific targeting of the conjugated modifying agent.

SPAs have been paired with the DNA-alkylating moieties cyclopropylpyrroloindole^[19] and chlorambucil^[20] that were able to damage and crosslink SV40 DNA. This effect inhibited cell cycling and growth. Chlorambucil, a chemotherapeutic agent, was more effective when conjugated to an SPA than without.

In 2012, SPAs were conjugated to SAHA, a potent histone deacetylase (HDAC) inhibitor.^[21] SPAs with conjugated SAHA were targeted to Oct-3/4 and Nanog which induced epigenetic remodeling and consequently increased expression of multiple pluripotency related genes in mouse embryonic fibroblasts.

Designer zinc-finger proteins

What they are/structure

Designer zinc-finger proteins are engineered proteins used to target specific areas of DNA. These proteins capitalize on the DNA-binding capacity of natural zinc-finger domains to modulate specific target areas of the genome.^[22] In both designer and natural zinc-finger motifs, the protein consists of two β-sheets and one α-helix. Two histidine residues on the α-helix and two cysteine residues on the β-sheets are bonded to a zinc atom, which serves to stabilize the protein domain as a whole. This stabilization particularly benefits the α-helix in its function as the DNA-recognition and -binding domain. Transcription factor TFIIIA is an example of a naturally-occurring protein with zinc-finger motifs.^[23]

Example of zinc-finger motifs

How they work

Zinc-finger motifs bind into the major groove of helical DNA,^[23] where the amino acid residue sequence on the α-helix gives the motif its target sequence specificity. The domain binds to a seven-nucleotide sequence of DNA (positions 1 through 6 on the primary strand of DNA, plus positions 0 and 3 on the complementary strand), thereby ensuring that the protein motif is highly selective of its target.^[22] In engineering a designer zinc-finger protein, researchers can utilize techniques such as site-directed mutagenesis followed by randomized trials for binding capacity,^[22][24] or the in vitro recombination of motifs with known target specificity to produce a library of sequence-specific final proteins.^[25]

Zinc fingers binding a DNA helix

 

Epigenetic mechanisms

Effects and impacts on gene modulation

Designer zinc-finger proteins can modulate genome expression in a number of ways. Ultimately, two factors are primarily responsible for the end result on expression: whether the targeted sequence is a regulatory region or a coding region of DNA, and whether and what types of effector domains are bound to the zinc-finger domain. If the target sequence for an engineered designer protein is a regulatory domain - e.g., a promoter or a repressor of replication - the binding site for naturally-occurring transcription factors will be obscured, leading to a corresponding decrease or increase, respectively, in transcription for the associated gene.^[26] Similarly, if the target sequence is an exon, the designer zinc-finger will obscure the sequence from RNA polymerase transcription complexes, resulting in a truncated or otherwise nonfunctional gene product.^[22]

Effector domains bound to the zinc-finger can also have comparable effects. It is the function of these effector domains which are arguably the most important with respect to the use of designer zinc-finger proteins for therapeutic gene modulation. If a methylase domain is bound to the designer zinc-finger protein, when the zinc-finger protein binds to the target DNA sequence an increase in methylation state of DNA in that region will subsequently result. Transcription rates of genes so-affected will be reduced.^[27] Many of the effector domains function to modulate either the DNA directly - e.g. via methylation, cleaving,^[28] or recombination of the target DNA sequence^[29] - or by modulating its transcription rate - e.g. inhibiting transcription via repressor domains that block transcriptional machinery,^[30] promoting transcription with activation domains that recruit transcriptional machinery to the site,^[31] or histone- or other epigenetic-modification domains that affect chromatin state and the ability of transcriptional machinery to access the affected genes.^[32] Epigenetic modification is a major theme in determining varying expression levels for genes, as explained by the idea that how tightly-wound the DNA strand is - from histones at the local level up to chromatin at the chromosomal level - can influence the accessibility of sequences of DNA to transcription machinery, thereby influencing the rate at which it can be transcribed.^[23] If, instead of impacting the DNA strand directly, as described above, a designer zinc-finger protein instead affects epigenetic modification state for a target DNA region, modulation of gene expression could similarly be accomplished.

In the first case to successfully demonstrate the use of designer zinc-finger proteins to modulate gene expression in vivo, Choo et al^[26] designed a protein consisting of three zinc-finger domains that targeted a specific sequence on a BCR-ABL fusion oncogene. This specific oncogene is implicated in acute lymphoblastic leukemia. The oncogene typically enables leukemia cells to proliferate in the absence of specific growth factors, a hallmark of cancer. By including a nuclear localization signal with the tri-domain zinc-finger protein in order to facilitate binding of the protein to genomic DNA in the nucleus, Choo et al were able to demonstrate that their engineered protein could block transcription of the oncogene in vivo. Leukemia cells became dependent on regular growth factors, bringing the cell cycle back under the control of normal regulation.^[26]

Post-transcriptional gene modulation

The major approach to post-transcriptional gene modulation is via RNA interference (RNAi). The primary problem with using RNAi in gene modulation is drug delivery to target cells.^[33][34] RNAi gene modulation has been successfully applied to mice toward the treatment of a mouse model for inflammatory bowel disease.^[35] This treatment utilized liposome-based beta-7 integrin-targeted, stabilized nanoparticles entrapping short interfering RNAs (siRNAs). There are several other forms of RNAi delivery, including: polyplex delivery, ligand-siRNA conjugates, naked delivery, inorganic particle deliver using gold nanoparticles, and site specific local delivery.^[36]

Clinical significance

Designer zinc-finger proteins, on the other hand, have undergone some trials in the clinical arena. The efficacy and safety of EW-A-401, an engineered zinc-finger transcription factor, as a pharmacologic agent for treating claudication, a cardiovascular ailment, has been investigated in clinical trials.^[37] The protein consists of an engineered plasmid DNA that prompts the patient to produce an engineered transcription factor, the target of which is the vascular endothelial growth factor-A (VEGF-A) gene, which positively influences blood vessel development. Although not yet approved by the U.S. Food and Drug Administration (FDA), two Phase I clinical studies have been completed which identify this zinc-finger protein as a promising and safe potential therapeutic agent for treatment of peripheral arterial disease in humans.

Gene therapy

From Wikipedia, the free encyclopedia

 

Gene therapy using an adenovirus vector. In some cases, the adenovirus will insert the new gene into a cell. If the treatment is successful, the new gene will make a functional protein to treat a disease.

In the medicine field, gene therapy (also called human gene transfer) is the therapeutic delivery of nucleic acid into a patient's cells as a drug to treat disease. The first attempt at modifying human DNA was performed in 1980 by Martin Cline, but the first successful nuclear gene transfer in humans, approved by the National Institutes of Health, was performed in May 1989. The first therapeutic use of gene transfer as well as the first direct insertion of human DNA into the nuclear genome was performed by French Anderson in a trial starting in September 1990.

Between 1989 and February 2016, over 2,300 clinical trials had been conducted, more than half of them in phase I.

Not all medical procedures that introduce alterations to a patient's genetic makeup can be considered gene therapy. Bone marrow transplantation and organ transplants in general have been found to introduce foreign DNA into patients.^[5] Gene therapy is defined by the precision of the procedure and the intention of direct therapeutic effects.

Background

Gene therapy was conceptualized in 1972, by authors who urged caution before commencing human gene therapy studies.

The first attempt, an unsuccessful one, at gene therapy (as well as the first case of medical transfer of foreign genes into humans not counting organ transplantation) was performed by Martin Cline on 10 July 1980.^[6][7] Cline claimed that one of the genes in his patients was active six months later, though he never published this data or had it verified^[8] and even if he is correct, it's unlikely it produced any significant beneficial effects treating beta-thalassemia.

After extensive research on animals throughout the 1980s and a 1989 bacterial gene tagging trial on humans, the first gene therapy widely accepted as a success was demonstrated in a trial that started on 14 September 1990, when Ashi DeSilva was treated for ADA-SCID.^[9]

The first somatic treatment that produced a permanent genetic change was performed in 1993.^{[citation needed]}

Gene therapy is a way to fix a genetic problem at its source. The polymers are either translated into proteins, interfere with target gene expression, or possibly correct genetic mutations.

The most common form uses DNA that encodes a functional, therapeutic gene to replace a mutated gene. The polymer molecule is packaged within a "vector", which carries the molecule inside cells.

Early clinical failures led to dismissals of gene therapy. Clinical successes since 2006 regained researchers' attention, although as of 2014, it was still largely an experimental technique.^[10] These include treatment of retinal diseases Leber's congenital amaurosis^{[11][12][13][14]} and choroideremia,^[15] X-linked SCID,^[16] ADA-SCID,^[17][18] adrenoleukodystrophy,^[19] chronic lymphocytic leukemia (CLL),^[20] acute lymphocytic leukemia (ALL),^[21] multiple myeloma,^[22] haemophilia,^[18] and Parkinson's disease.^[23] Between 2013 and April 2014, US companies invested over $600 million in the field.^[24]

The first commercial gene therapy, Gendicine, was approved in China in 2003 for the treatment of certain cancers.^[25] In 2011 Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease, including critical limb ischemia.^[26] In 2012 Glybera, a treatment for a rare inherited disorder, became the first treatment to be approved for clinical use in either Europe or the United States after its endorsement by the European Commission.^[10][27]

Approaches

Following early advances in genetic engineering of bacteria, cells, and small animals, scientists started considering how to apply it to medicine. Two main approaches were considered – replacing or disrupting defective genes.^[28] Scientists focused on diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy, thalassemia, and sickle cell anemia. Glybera treats one such disease, caused by a defect in lipoprotein lipase.^[27]

DNA must be administered, reach the damaged cells, enter the cell and either express or disrupt a protein.^[29] Multiple delivery techniques have been explored. The initial approach incorporated DNA into an engineered virus to deliver the DNA into a chromosome.^[30][31] Naked DNA approaches have also been explored, especially in the context of vaccine development.^[32]

Generally, efforts focused on administering a gene that causes a needed protein to be expressed. More recently, increased understanding of nuclease function has led to more direct DNA editing, using techniques such as zinc finger nucleases and CRISPR. The vector incorporates genes into chromosomes. The expressed nucleases then knock out and replace genes in the chromosome. As of 2014 these approaches involve removing cells from patients, editing a chromosome and returning the transformed cells to patients.^[33]

Gene editing is a potential approach to alter the human genome to treat genetic diseases,^[34] viral diseases,^[35] and cancer.^[36] As of 2016 these approaches were still years from being medicine.^[37][38]
 

A duplex of crRNA and tracrRNA acts as guide RNA to

introduce a specifically located gene modification based on

the RNA 5’ upstream of the crRNA. Cas9 binds the tracrRNA

and needs a DNA binding sequence (5’NGG3’), which is

called protospacer adjacent motif (PAM). After binding, Cas9

introduces a DNA double strand break, which is then followed

by gene modification via homologous recombination (HDR) or

non-homologous end joining (NHEJ).

Cell types

Gene therapy may be classified into two types:

Somatic

In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Any such modifications affect the individual patient only, and are not inherited by offspring. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease.

Over 600 clinical trials utilizing SCGT are underway in the US. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia, and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy. The complete correction of a genetic disorder or the replacement of multiple genes is not yet possible. Only a few of the trials are in the advanced stages.^[39]

Germline

In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene. The change is therefore heritable and passed on to later generations. Australia, Canada, Germany, Israel, Switzerland, and the Netherlands^[40] prohibit GGT for application in human beings, for technical and ethical reasons, including insufficient knowledge about possible risks to future generations^[40] and higher risks versus SCGT.^[41] The US has no federal controls specifically addressing human genetic modification (beyond FDA regulations for therapies in general).^{[40][42][43][44]}

Vectors

The delivery of DNA into cells can be accomplished by multiple methods. The two major classes are recombinant viruses (sometimes called biological nanoparticles or viral vectors) and naked DNA or DNA complexes (non-viral methods).

Viruses

In order to replicate, viruses introduce their genetic material into the host cell, tricking the host's cellular machinery into using it as blueprints for viral proteins. Retroviruses go a stage further by having their genetic material copied into the genome of the host cell. Scientists exploit this by substituting a virus's genetic material with therapeutic DNA. (The term 'DNA' may be an oversimplification, as some viruses contain RNA, and gene therapy could take this form as well.) A number of viruses have been used for human gene therapy, including retroviruses, adenoviruses, herpes simplex, vaccinia, and adeno-associated virus.^[4] Like the genetic material (DNA or RNA) in viruses, therapeutic DNA can be designed to simply serve as a temporary blueprint that is degraded naturally or (at least theoretically) to enter the host's genome, becoming a permanent part of the host's DNA in infected cells.

Non-viral

Non-viral methods present certain advantages over viral methods, such as large scale production and low host immunogenicity. However, non-viral methods initially produced lower levels of transfection and gene expression, and thus lower therapeutic efficacy. Later technology remedied this deficiency.

Methods for non-viral gene therapy include the injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.

Hurdles

Some of the unsolved problems include:

• Short-lived nature – Before gene therapy can become a permanent cure for a condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent it from achieving long-term benefits. Patients require multiple treatments.
• Immune response – Any time a foreign object is introduced into human tissues, the immune system is stimulated to attack the invader. Stimulating the immune system in a way that reduces gene therapy effectiveness is possible. The immune system's enhanced response to viruses that it has seen before reduces the effectiveness to repeated treatments.
• Problems with viral vectors – Viral vectors carry the risks of toxicity, inflammatory responses, and gene control and targeting issues.
• Multigene disorders – Some commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are affected by variations in multiple genes, which complicate gene therapy.
• Some therapies may breach the Weismann barrier (between soma and germ-line) protecting the testes, potentially modifying the germline, falling afoul of regulations in countries that prohibit the latter practice.^[45]
• Insertional mutagenesis – If the DNA is integrated in a sensitive spot in the genome, for example in a tumor suppressor gene, the therapy could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.^[46][47] One possible solution is to add a functional tumor suppressor gene to the DNA to be integrated. This may be problematic since the longer the DNA is, the harder it is to integrate into cell genomes. CRISPR technology allows researchers to make much more precise genome changes at exact locations.^[48]
• Cost – Alipogene tiparvovec or Glybera, for example, at a cost of $1.6 million per patient, was reported in 2013 to be the world's most expensive drug.^[49][50]

Deaths

Three patients' deaths have been reported in gene therapy trials, putting the field under close scrutiny. The first was that of Jesse Gelsinger in 1999. Jesse Gelsinger died because of immune rejection response.^[51] One X-SCID patient died of leukemia in 2003.^[9] In 2007, a rheumatoid arthritis patient died from an infection; the subsequent investigation concluded that the death was not related to gene therapy.^[52]

History

1970s and earlier

In 1972 Friedmann and Roblin authored a paper in Science titled "Gene therapy for human genetic disease?"^[53] Rogers (1970) was cited for proposing that exogenous good DNA be used to replace the defective DNA in those who suffer from genetic defects.^[54]

1980s

In 1984 a retrovirus vector system was designed that could efficiently insert foreign genes into mammalian chromosomes.^[55]

1990s

The first approved gene therapy clinical research in the US took place on 14 September 1990, at the National Institutes of Health (NIH), under the direction of William French Anderson.^[56] Four-year-old Ashanti DeSilva received treatment for a genetic defect that left her with ADA-SCID, a severe immune system deficiency. The defective gene of the patient's blood cells was replaced by the functional variant. Ashanti’s immune system was partially restored by the therapy. Production of the missing enzyme was temporarily stimulated, but the new cells with functional genes were not generated. She led a normal life only with the regular injections performed every two months. The effects were successful, but temporary.^[57]

Cancer gene therapy was introduced in 1992/93 (Trojan et al. 1993).^[58] The treatment of glioblastoma multiforme, the malignant brain tumor whose outcome is always fatal, was done using a vector expressing antisense IGF-I RNA (clinical trial approved by NIH protocolno.1602 November 24, 1993,^[59] and by the FDA in 1994). This therapy also represents the beginning of cancer immunogene therapy, a treatment which proves to be effective due to the anti-tumor mechanism of IGF-I antisense, which is related to strong immune and apoptotic phenomena.

In 1992 Claudio Bordignon, working at the Vita-Salute San Raffaele University, performed the first gene therapy procedure using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases.^[60] In 2002 this work led to the publication of the first successful gene therapy treatment for adenosine deaminase deficiency (ADA-SCID). The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) from 2000 and 2002, was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the US, the United Kingdom, France, Italy, and Germany.^[61]

In 1993 Andrew Gobea was born with SCID following prenatal genetic screening. Blood was removed from his mother's placenta and umbilical cord immediately after birth, to acquire stem cells. The allele that codes for adenosine deaminase (ADA) was obtained and inserted into a retrovirus. Retroviruses and stem cells were mixed, after which the viruses inserted the gene into the stem cell chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood. Injections of the ADA enzyme were also given weekly. For four years T cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed.^[62]

Jesse Gelsinger's death in 1999 impeded gene therapy research in the US.^[63][64] As a result, the FDA suspended several clinical trials pending the reevaluation of ethical and procedural practices.^[65]

2000s

The modified cancer gene therapy strategy of antisense IGF-I RNA (NIH n˚ 1602)^[59] using antisense / triple helix anti-IGF-I approach was registered in 2002 by Wiley gene therapy clinical trial - n˚ 635 and 636. The approach has shown promising results in the treatment of six different malignant tumors: glioblastoma, cancers of liver, colon, prostate, uterus, and ovary (Collaborative NATO Science Programme on Gene Therapy USA, France, Poland n˚ LST 980517 conducted by J. Trojan) (Trojan et al., 2012). This anti-gene antisense/triple helix therapy has proven to be efficient, due to the mechanism stopping simultaneously IGF-I expression on translation and transcription levels, strengthening anti-tumor immune and apoptotic phenomena.

2002

Sickle-cell disease can be treated in mice.^[66] The mice – which have essentially the same defect that causes human cases – used a viral vector to induce production of fetal hemoglobin (HbF), which normally ceases to be produced shortly after birth. In humans, the use of hydroxyurea to stimulate the production of HbF temporarily alleviates sickle cell symptoms. The researchers demonstrated this treatment to be a more permanent means to increase therapeutic HbF production.^[67]

A new gene therapy approach repaired errors in messenger RNA derived from defective genes. This technique has the potential to treat thalassaemia, cystic fibrosis and some cancers.^[68]

Researchers created liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane.^[69]

2003

In 2003 a research team inserted genes into the brain for the first time. They used liposomes coated in a polymer called polyethylene glycol, which unlike viral vectors, are small enough to cross the blood–brain barrier.^[70]

Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced.^[71]

Gendicine is a cancer gene therapy that delivers the tumor suppressor gene p53 using an engineered adenovirus. In 2003, it was approved in China for the treatment of head and neck squamous cell carcinoma.^[25]

2006

In March researchers announced the successful use of gene therapy to treat two adult patients for X-linked chronic granulomatous disease, a disease which affects myeloid cells and damages the immune system. The study is the first to show that gene therapy can treat the myeloid system.^[72]

In May a team reported a way to prevent the immune system from rejecting a newly delivered gene.^[73] Similar to organ transplantation, gene therapy has been plagued by this problem. The immune system normally recognizes the new gene as foreign and rejects the cells carrying it. The research utilized a newly uncovered network of genes regulated by molecules known as microRNAs. This natural function selectively obscured their therapeutic gene in immune system cells and protected it from discovery. Mice infected with the gene containing an immune-cell microRNA target sequence did not reject the gene.

In August scientists successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells.^[74]

In November researchers reported on the use of VRX496, a gene-based immunotherapy for the treatment of HIV that uses a lentiviral vector to deliver an antisense gene against the HIV envelope. In a phase I clinical trial, five subjects with chronic HIV infection who had failed to respond to at least two antiretroviral regimens were treated. A single intravenous infusion of autologous CD4 T cells genetically modified with VRX496 was well tolerated. All patients had stable or decreased viral load; four of the five patients had stable or increased CD4 T cell counts. All five patients had stable or increased immune response to HIV antigens and other pathogens. This was the first evaluation of a lentiviral vector administered in a US human clinical trial.^[75][76]

2007

In May researchers announced the first gene therapy trial for inherited retinal disease. The first operation was carried out on a 23-year-old British male, Robert Johnson, in early 2007.^[77]

2008

Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of a small clinical trial in children were published in April.^[11] Delivery of recombinant adeno-associated virus (AAV) carrying RPE65 yielded positive results. In May two more groups reported positive results in independent clinical trials using gene therapy to treat the condition. In all three clinical trials, patients recovered functional vision without apparent side-effects.^{[11][12][13][14]}

2009

In September researchers were able to give trichromatic vision to squirrel monkeys.^[78] In November 2009, researchers halted a fatal genetic disorder called adrenoleukodystrophy in two children using a lentivirus vector to deliver a functioning version of ABCD1, the gene that is mutated in the disorder.^[79]

2010s

2010

An April paper reported that gene therapy addressed achromatopsia (color blindness) in dogs by targeting cone photoreceptors. Cone function and day vision were restored for at least 33 months in two young specimens. The therapy was less efficient for older dogs.^[80]

In September it was announced that an 18-year-old male patient in France with beta-thalassemia major had been successfully treated.^[81] Beta-thalassemia major is an inherited blood disease in which beta haemoglobin is missing and patients are dependent on regular lifelong blood transfusions.^[82] The technique used a lentiviral vector to transduce the human ß-globin gene into purified blood and marrow cells obtained from the patient in June 2007.^[83] The patient's haemoglobin levels were stable at 9 to 10 g/dL. About a third of the hemoglobin contained the form introduced by the viral vector and blood transfusions were not needed.^[83][84] Further clinical trials were planned.^[85] Bone marrow transplants are the only cure for thalassemia, but 75% of patients do not find a matching donor.^[84]

Cancer immunogene therapy using modified antigene, antisense/triple helix approach was introduced in South America in 2010/11 in La Sabana University, Bogota (Ethical Committee 14 December 2010, no P-004-10). Considering the ethical aspect of gene diagnostic and gene therapy targeting IGF-I, the IGF-I expressing tumors i.e. lung and epidermis cancers were treated (Trojan et al. 2016).

2011

In 2007 and 2008, a man (Timothy Ray Brown) was cured of HIV by repeated hematopoietic stem cell transplantation (see also allogeneic stem cell transplantation, allogeneic bone marrow transplantation, allotransplantation) with double-delta-32 mutation which disables the CCR5 receptor. This cure was accepted by the medical community in 2011.^[88] It required complete ablation of existing bone marrow, which is very debilitating.

In August two of three subjects of a pilot study were confirmed to have been cured from chronic lymphocytic leukemia (CLL). The therapy used genetically modified T cells to attack cells that expressed the CD19 protein to fight the disease.^[20] In 2013, the researchers announced that 26 of 59 patients had achieved complete remission and the original patient had remained tumor-free.^[89]

Human HGF plasmid DNA therapy of cardiomyocytes is being examined as a potential treatment for coronary artery disease as well as treatment for the damage that occurs to the heart after myocardial infarction.^[90][91]

In 2011 Neovasculgen was registered in Russia as the first-in-class gene-therapy drug for treatment of peripheral artery disease, including critical limb ischemia; it delivers the gene encoding for VEGF.^[92][26] Neovasculogen is a plasmid encoding the CMV promoter and the 165 amino acid form of VEGF.^[93][94]

2012

The FDA approved Phase 1 clinical trials on thalassemia major patients in the US for 10 participants in July.^[95] The study was expected to continue until 2015.^[85]

In July 2012, the European Medicines Agency recommended approval of a gene therapy treatment for the first time in either Europe or the United States. The treatment used Alipogene tiparvovec (Glybera) to compensate for lipoprotein lipase deficiency, which can cause severe pancreatitis.^[96] The recommendation was endorsed by the European Commission in November 2012^{[10][27][97][98]} and commercial rollout began in late 2014.^[99] Alipogene tiparvovec was expected to cost around $1.6 million per treatment in 2012,^[100] revised to $1 million in 2015,^[101] making it the most expensive medicine in the world at the time.^[102] As of 2016, only one person had been treated with drug.^[103]

In December 2012, it was reported that 10 of 13 patients with multiple myeloma were in remission "or very close to it" three months after being injected with a treatment involving genetically engineered T cells to target proteins NY-ESO-1 and LAGE-1, which exist only on cancerous myeloma cells.^[22]

2013

In March researchers reported that three of five adult subjects who had acute lymphocytic leukemia (ALL) had been in remission for five months to two years after being treated with genetically modified T cells which attacked cells with CD19 genes on their surface, i.e. all B-cells, cancerous or not. The researchers believed that the patients' immune systems would make normal T-cells and B-cells after a couple of months. They were also given bone marrow. One patient relapsed and died and one died of a blood clot unrelated to the disease.^[21]

Following encouraging Phase 1 trials, in April, researchers announced they were starting Phase 2 clinical trials (called CUPID2 and SERCA-LVAD) on 250 patients^[104] at several hospitals to combat heart disease. The therapy was designed to increase the levels of SERCA2, a protein in heart muscles, improving muscle function.^[105] The FDA granted this a Breakthrough Therapy Designation to accelerate the trial and approval process.^[106] In 2016 it was reported that no improvement was found from the CUPID 2 trial.^[107]

In July researchers reported promising results for six children with two severe hereditary diseases had been treated with a partially deactivated lentivirus to replace a faulty gene and after 7–32 months. Three of the children had metachromatic leukodystrophy, which causes children to lose cognitive and motor skills.^[108] The other children had Wiskott-Aldrich syndrome, which leaves them to open to infection, autoimmune diseases, and cancer.^[109] Follow up trials with gene therapy on another six children with Wiskott-Aldrich syndrome were also reported as promising.^[110][111]

In October researchers reported that two children born with adenosine deaminase severe combined immunodeficiency disease (ADA-SCID) had been treated with genetically engineered stem cells 18 months previously and that their immune systems were showing signs of full recovery. Another three children were making progress.^[18] In 2014 a further 18 children with ADA-SCID were cured by gene therapy.^[112] ADA-SCID children have no functioning immune system and are sometimes known as "bubble children."^[18]

Also in October researchers reported that they had treated six hemophilia sufferers in early 2011 using an adeno-associated virus. Over two years later all six were producing clotting factor.^[18][113]

2014

In January researchers reported that six choroideremia patients had been treated with adeno-associated virus with a copy of REP1. Over a six-month to two-year period all had improved their sight.^[114][115] By 2016, 32 patients had been treated with positive results and researchers were hopeful the treatment would be long-lasting.^[15] Choroideremia is an inherited genetic eye disease with no approved treatment, leading to loss of sight.

In March researchers reported that 12 HIV patients had been treated since 2009 in a trial with a genetically engineered virus with a rare mutation (CCR5 deficiency) known to protect against HIV with promising results.^[116][117]

Clinical trials of gene therapy for sickle cell disease were started in 2014.^[118][119] There is a need for high quality randomised controlled trials assessing the risks and benefits involved with gene therapy for people with sickle cell disease.^[120]

2015

In February LentiGlobin BB305, a gene therapy treatment undergoing clinical trials for treatment of beta thalassemia gained FDA "breakthrough" status after several patients were able to forgo the frequent blood transfusions usually required to treat the disease.^[121]

In March researchers delivered a recombinant gene encoding a broadly neutralizing antibody into monkeys infected with simian HIV; the monkeys' cells produced the antibody, which cleared them of HIV. The technique is named immunoprophylaxis by gene transfer (IGT). Animal tests for antibodies to ebola, malaria, influenza, and hepatitis were underway.^[122][123]

In March, scientists, including an inventor of CRISPR, Jennifer Doudna, urged a worldwide moratorium on germline gene therapy, writing "scientists should avoid even attempting, in lax jurisdictions, germline genome modification for clinical application in humans" until the full implications "are discussed among scientific and governmental organizations".^{[124][125][126][127]}

In October, researchers announced that they had treated a baby girl, Layla Richards, with an experimental treatment using donor T-cells genetically engineered using TALEN to attack cancer cells. One year after the treatment she was still free of her cancer (a highly aggressive form of acute lymphoblastic leukaemia [ALL]).^[128] Children with highly aggressive ALL normally have a very poor prognosis and Layla's disease had been regarded as terminal before the treatment.^[129]

In December, scientists of major world academies called for a moratorium on inheritable human genome edits, including those related to CRISPR-Cas9 technologies^[130] but that basic research including embryo gene editing should continue.^[131]

2016

In April the Committee for Medicinal Products for Human Use of the European Medicines Agency endorsed a gene therapy treatment called Strimvelis^[132][133] and the European Commission approved it in June.^[134] This treats children born with adenosine deaminase deficiency and who have no functioning immune system. This was the second gene therapy treatment to be approved in Europe.^[135]

In October, Chinese scientists reported they had started a trial to genetically modify T-cells from 10 adult patients with lung cancer and reinject the modified T-cells back into their bodies to attack the cancer cells. The T-cells had the PD-1 protein (which stops or slows the immune response) removed using CRISPR-Cas9.^[136][137]

A 2016 Cochrane systematic review looking at data from four trials on topical cystic fibrosis transmembrane conductance regulator (CFTR) gene therapy does not support its clinical use as a mist inhaled into the lungs to treat cystic fibrosis patients with lung infections. One of the four trials did find weak evidence that liposome-based CFTR gene transfer therapy may lead to a small respiratory improvement for people with CF. This weak evidence is not enough to make a clinical recommendation for routine CFTR gene therapy.^[138]

2017

In February Kite Pharma announced results from a clinical trial of CAR-T cells in around a hundred people with advanced Non-Hodgkin lymphoma.^[139]

In March, French scientists reported on clinical research of gene therapy to treat sickle-cell disease.^[140]

In August, the FDA approved tisagenlecleucel for acute lymphoblastic leukemia.^[141] Tisagenlecleucel is an adoptive cell transfer therapy for B-cell acute lymphoblastic leukemia; T cells from a person with cancer are removed, genetically engineered to make a specific T-cell receptor (a chimeric T cell receptor, or "CAR-T") that reacts to the cancer, and are administered back to the person. The T cells are engineered to target a protein called CD19 that is common on B cells. This is the first form of gene therapy to be approved in the United States. In October, a similar therapy called axicabtagene ciloleucel was approved for non-Hodgkin lymphoma.^[142]

In December the results of using an adeno-associated virus with blood clotting factor VIII to treat nine haemophilia A patients were published. Six of the seven patients on the high dose regime increased the level of the blood clotting VIII to normal levels. The low and medium dose regimes had no effect on the patient's blood clotting levels.^[143][144]

In December, the FDA approved Luxturna, the first in vivo gene therapy, for the treatment of blindness due to Leber's congenital amaurosis.^[145] The price of this treatment was 850,000 US dollars for both eyes.^[146][147]

Speculative uses

Speculated uses for gene therapy include:

Fertility

Gene Therapy techniques have the potential to provide alternative treatments for those with infertility. Recently, successful experimentation on mice has proven that fertility can be restored by using the gene therapy method, CRISPR.^[148] Spermatogenical stem cells from another organism were transplanted into the testes of an infertile male mouse. The stem cells re-established spermatogenesis and fertility.^[149]

Gene doping

Athletes might adopt gene therapy technologies to improve their performance.^[150] Gene doping is not known to occur, but multiple gene therapies may have such effects. Kayser et al. argue that gene doping could level the playing field if all athletes receive equal access. Critics claim that any therapeutic intervention for non-therapeutic/enhancement purposes compromises the ethical foundations of medicine and sports.^[151]

Human genetic engineering

Genetic engineering could be used to cure diseases, but also to change physical appearance, metabolism, and even improve physical capabilities and mental faculties such as memory and intelligence. Ethical claims about germline engineering include beliefs that every fetus has a right to remain genetically unmodified, that parents hold the right to genetically modify their offspring, and that every child has the right to be born free of preventable diseases.^{[152][153][154]} For parents, genetic engineering could be seen as another child enhancement technique to add to diet, exercise, education, training, cosmetics, and plastic surgery.^[155][156] Another theorist claims that moral concerns limit but do not prohibit germline engineering.^[157]
Possible regulatory schemes include a complete ban, provision to everyone, or professional self-regulation. The American Medical Association’s Council on Ethical and Judicial Affairs stated that "genetic interventions to enhance traits should be considered permissible only in severely restricted situations: (1) clear and meaningful benefits to the fetus or child; (2) no trade-off with other characteristics or traits; and (3) equal access to the genetic technology, irrespective of income or other socioeconomic characteristics."^[158]

As early in the history of biotechnology as 1990, there have been scientists opposed to attempts to modify the human germline using these new tools,^[159] and such concerns have continued as technology progressed.^[160][161] With the advent of new techniques like CRISPR, in March 2015 a group of scientists urged a worldwide moratorium on clinical use of gene editing technologies to edit the human genome in a way that can be inherited.^{[124][125][126][127]} In April 2015, researchers sparked controversy when they reported results of basic research to edit the DNA of non-viable human embryos using CRISPR.^[148][162] A committee of the American National Academy of Sciences and National Academy of Medicine gave qualified support to human genome editing in 2017^[163][164] once answers have been found to safety and efficiency problems "but only for serious conditions under stringent oversight."^[165]

Regulations

Regulations covering genetic modification are part of general guidelines about human-involved biomedical research. There are no international treaties which are legally binding in this area, but there are recommendations for national laws from various bodies.

The Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects) was amended by the World Medical Association's General Assembly in 2008. This document provides principles physicians and researchers must consider when involving humans as research subjects. The Statement on Gene Therapy Research initiated by the Human Genome Organization (HUGO) in 2001 provides a legal baseline for all countries. HUGO’s document emphasizes human freedom and adherence to human rights, and offers recommendations for somatic gene therapy, including the importance of recognizing public concerns about such research.^[166]

United States

No federal legislation lays out protocols or restrictions about human genetic engineering. This subject is governed by overlapping regulations from local and federal agencies, including the Department of Health and Human Services, the FDA and NIH's Recombinant DNA Advisory Committee. Researchers seeking federal funds for an investigational new drug application, (commonly the case for somatic human genetic engineering,) must obey international and federal guidelines for the protection of human subjects.^[167]

NIH serves as the main gene therapy regulator for federally funded research. Privately funded research is advised to follow these regulations. NIH provides funding for research that develops or enhances genetic engineering techniques and to evaluate the ethics and quality in current research. The NIH maintains a mandatory registry of human genetic engineering research protocols that includes all federally funded projects.

An NIH advisory committee published a set of guidelines on gene manipulation.^[168] The guidelines discuss lab safety as well as human test subjects and various experimental types that involve genetic changes. Several sections specifically pertain to human genetic engineering, including Section III-C-1. This section describes required review processes and other aspects when seeking approval to begin clinical research involving genetic transfer into a human patient.^[169] The protocol for a gene therapy clinical trial must be approved by the NIH's Recombinant DNA Advisory Committee prior to any clinical trial beginning; this is different from any other kind of clinical trial.^[168]

As with other kinds of drugs, the FDA regulates the quality and safety of gene therapy products and supervises how these products are used clinically. Therapeutic alteration of the human genome falls under the same regulatory requirements as any other medical treatment. Research involving human subjects, such as clinical trials, must be reviewed and approved by the FDA and an Institutional Review Board.^[170][171]

Popular culture

Gene therapy is the basis for the plotline of the film I Am Legend^[172] and the TV show Will Gene Therapy Change the Human Race?.^[173] In 1994, gene therapy was a plot element in The Erlenmeyer Flask, The X-Files' first season finale. It is also used in Stargate as a means of allowing humans to use Ancient technology.