Introduction to gene therapy

Question 1

What is gene therapy?

Answer 1

•The replacement or alteration of a defective gene in order to treat a disorder caused by a mutation or deficiency in that gene.

–Addition of a functional gene

–Replacement of a faulty gene with a functional one

–Repair of a non-functional gene

–Alteration of expression of a gene

However, the potential scope of gene therapy also extends to the prevention of infectious diseases and the customisation of embryos prior to implantation. As technology and society advances, the definition of what is and isn’t ethically acceptable is likely to change

Question 2

What is an important consideration for gene therapy?

Answer 2

• An important consideration is whether the treatment is temporary (transient expression) or permanent (stable transfection).
• A transient treatment will require regular repeats whereas a permanent treatment cannot easily be reversed

Question 3

What are the strategies for gee therapy?

Answer 3

There are several strategies:

• by addition of a functional gene to complement a deficiency in a gene product. This is the simplest to perform and is by far the simplest and most common approach. The gene, under the control of its own promoter (which may be constitutively-expressing, tissue-specific or inducible), is delivered into cells and may integrate into a random area of the host cell genome (for example a gamma retrovirus, γ-RV) or persist as episomal DNA in the nucleus (for example delivery by adeno-associated virus, AAV).
• by replacement of a faulty gene with a functional copy. This is exchange of the faulty gene or part of the gene by homologous recombination with a functional copy and relies on targeting by surrounding homologous DNA sequences and requires the host cell DNA recombination systems. This method has been used successfully to modify mouse genomes in the past. The scientists responsible for its discovery were awarded the 2007 Nobel prize in Physiology or Medicine.
• By selective repair of a non-functional gene. This approach involves targeted nuclease based gene editing technologies such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat – Cas associated nucleases (CRISPR-Cas9).
• By altering the expression level of a gene, a reduction in the amount of a protein involved in a disease process can be achieved, for example by the use of RNA interference. It is also possible to alter how an mRNA is spliced, for example to selectively remove an exon containing a damaging mutation by masking a splice signal with a complementary synthetic oligonucleotide.

Question 4

Show a graph of the approved gene therapy clinical trials worldwide

Answer 4

• This chart shows the number of approved gene therapy clinical trials around the world.
• Data is not current because there is no central database for gene therapy trials.
• Data is compiled mostly from publications and so is subject to some delay.
• There has been a steady increase in trials of gene therapies since the first trial in 1989, which reflects the continued drive to develop new technologies

Question 5

There are some notable dips in the data and these occur immediately after some of the setbacks that highlighted safety issues with gene therapy.

Give some examples of why this is

Answer 5

• For example, the first death caused by gene therapy occurred in 1999 due to an immune response to an adenoviral vector (Jesse Gelsinger).
• In 2007, reports emerged about the development of leukaemia in four babies treated using a gamma retroviral vector for X-linked severe combined immunodeficiency (SCID-X1), with one subsequent death.
• This prompted a reconsideration of the safety of these viruses for human gene therapy trials – it also stimulated research into novel solutions to the problem of retrovirus insertional oncogenesis

Question 6

Show Gene therapy clinical trials by country

Answer 6

• Most gene therapy trials are conducted in the US, with China and the UK quite a way behind.
• A recent report (March 2020) suggests that there are currently 362 cell and gene therapies in clinical pipelines in the US.
• This probably reflects US domination in research, particularly in medicine.

Question 7

Why do we need gene therapy?

Answer 7

•Cure for untreatable or difficult to treat:

–Monogenic disorders

–Polygenic disorders

–Cancer

Question 8

Explain why we need gene therapy?

Answer 8

• Gene therapy is currently the last resort treatment for diseases and disorders that have no viable treatment options.
• Thousands of diseases and disorders are caused by a genetic mutation in a single gene.
• These mutations usually result in a loss of, or reduction in, protein functionality that manifests in a variety of different ways. For example, loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) in cystic fibrosis or loss of an enzyme activity in congenital metabolic disorders such as phenylketonuria.
• The multitude of different classes of mutations in CFTR is a perfect example of the power of gene therapy by addition of a functional gene to cells, rather than the very limited treatment options available for severe forms of CF. Many more diseases are the result of mutations in more than one gene.
• These are more difficult to treat because the interplay between genes is often complex and poorly understood.
• Cancer is itself a complex disease and gene therapies for cancer are designed to increase the host immune response to cancer cells or limit the proliferation of cancer cells and/or increase their susceptibility to chemotherapeutic drugs.

Question 9

Gene therapy clinical trials by disorder

Answer 9

• It is perhaps surprising that monogenic disorders are the subject of only 11.7% of gene therapy clinical trials.
• This is likely due to the lack of market demand – monogenic disorders are relatively rare and have been exploited by companies for proof-of-principle research.
• It is easier to obtain approval for human trials where the drug represents a last-resort treatment. Orphan drug status, which is used to promote research into rare diseases with very small markets, also gives significant financial and marketing benefits to a company.
• Unfortunately, very small markets result in very large costs per individual treatment, for example Zolgensma by Novartis costs 2.1 million dollars for a single dose treatment. Gene therapies for cancer are generic and can be applied to a large number of patients, explaining the disproportionate number of clinical trials into cancer therapies.

Question 10

Describe modes of treatment delivery

Answer 10

•In vivo

–Delivered directly into the body

–In situ

•Delivered directly to a particular organ or tissue

•Ex vivo

–Cells or tissues removed from the body, treated and then returned

Question 11

Explain modes of treatment delivery

Answer 11

• Gene therapies can be delivered to the body in a number of different ways. In vivo therapies are delivered directly into the body of the patient. This could be by injection into the bloodstream, the brain or the muscles for example. An in situ therapy is delivered directly to a particular target tissue. A good example would be inhalation of liposomes containing functional CFTR genes directly to the lung epithelia in cystic fibrosis patients. Ex vivo treatments involve removal of cells or tissue from the body of the patient, treatment with the gene therapy and return to the patient. A good example of an ex vivo gene therapy would be removal of CD34+ hematopoietic stem cells from the bone marrow, proliferation in vitro with cytokines and growth factors, treatment with a functional gene-containing retrovirus, and then reintroduction into the bone marrow of the patient.
• In vivo therapies are usually ones that need no particular tissue targeting, for example systemic expression of an enzyme to treat a congenital metabolic disorder. Otherwise, treatment of certain tissues can be achieved by the use of viruses that preferentially infect certain cell types or by the use of tissue-specific promoters. The in situ approach is most commonly used for tissue targeting since delivery can be more easily achieved in this way.

Question 12

When to use gene therapy?

Answer 12

•Germ line vs somatic?

–Germ line treatments are heritable through subsequent generations

–Somatic cell treatments are not heritable

•Adult, child or embryo?

–Informed consent?

• Disease vs functional enhancement?
• Risk vs reward?
• Acceptability to society?

Question 13

TRUE or FALSE: It is possible to use gene therapy to treat any type of cell in the human body.

Answer 13

TRUE

Question 14

Explain why It is possible to use gene therapy to treat any type of cell in the human body

Answer 14

The germ line cells give rise to the gametes and this genetic material is ultimately inherited from generation to generation. This raises an important ethical question about whether it is acceptable to make changes to all the future descendants of an individual. Do future descendants have a right to retrospectively question the decision of a perhaps deceased ancestor? There is also the question of long term cross-generation safety of gene therapies that are still in their early stages of development. This is one of the main driving forces behind the current legislation adopted by most countries that has banned germ line gene therapies. Any other cell in the body that isn’t a germ cell is a somatic cell, whose genetic material is not heritable. Only somatic cell therapies are currently legal and viewed as ethically acceptable by many.

Since any cell type can be a target for gene therapy, so can any developmental stage of an organism, from embryo to adult. An adult human patient is in a position to make a decision whether or not to undergo gene therapy based on expert advice from clinicians, counsellors and friends and family, so they are in a position of informed consent. However, many of the congenital disorders that are amenable to gene therapy treatment result in either premature abortion of the foetus, or manifest in early life with a rapid progression of symptoms resulting in irreversible damage or death, well before adulthood. Parents or legal guardians are able to make healthcare decisions for children below the age of consent. In the case of congenital disorders, this is often necessary to allow timely and effective treatment. In the case of in vitro fertilisation, embryos are already routinely screened for genetic defects and are selected for implantation on that basis. For any embryo there is an opportunity to screen for and potentially repair other significant genetic defects, particularly if both parents are carriers of a congenital disorder. Although not currently ethically acceptable or legal, it may be possible in the future to screen for other desirable characteristics, such as height, strength or intelligence and to edit the genome to enhance these characteristics.

For any gene therapy, there is an element of risk associated with the process which needs to be considered in light of the benefits of the gene therapy versus existing treatments, where available. In some cases a disorder can be managed to varying degrees with alternative treatments and this may be the best option. Other disorders are inevitably terminal and gene therapy is the only treatment, despite any risks.

The gauge of acceptability is what is generally tolerated by society, which can vary in different cultures and is liable to change with time and advances in technology.

Question 15

What are the vectors for delivery of genes?

Answer 15

•Naked plasmid DNA

–Poor uptake

–Poor expression

•Encapsulated plasmid DNA

–More stable in the body

–Better uptake

•Viruses

–Highly efficient at infection

–Can integrate into the genome

–Engineered to be replication-incompetent

Question 16

Gene therapies can be delivered by a variety of methods.

Explain them

Answer 16

• The simplest is to add plasmid DNA to cells – some of these plasmids will be taken up by the cells and find their way to the nucleus where they can express an encoded gene.
• This is inefficient because DNA is not particularly stable, especially in the body, and uptake into cells is poor. These factors together lead to poor expression and this method has not had much success.
• A better method is to encapsulate the DNA by incorporation into liposomes, which protects the DNA whilst in the body and promotes uptake into cells. This method has had successes for in situ gene therapies such as cystic fibrosis.
• Interestingly, this is the same method that the recently discovered Covid-19 vaccines use – mRNA encoding the spike protein inside liposomes is delivered by injection into muscle and the transient expression of spike protein in muscle cells results in a protective immune response. By far the most common vectors for delivery of DNA into cells are viruses.
• These have evolved highly efficient mechanisms for binding specifically to their host cells and inserting their genetic material inside. Some of them are able to integrate copies of their genetic material into the host genome to confer a permanent change. When viruses are used for gene therapy, viral genes that are required for replication are removed so that they remain infectious but are replication-incompetent.

Question 17

What are the basic properties of viral vectors?

Answer 17

•Adenoviruses (AVs)

–Respiratory infections

–dsDNA genome 26 – 46 kb

–Do not integrate (safer)

–Lost during cell replication by dilution (transient)

–Can elicit immune responses

•Modern varieties are engineered

Question 18

What are Adenoviruse?

Answer 18

• a group of dsDNA viruses that typically infect epithelia and mostly cause respiratory infections, such as the common cold.
• They can also cause eye and intestinal infections. With genomes ranging from 26 kilobases up to 46 kilobases, they have a large potential capacity for foreign DNA. Adenovirus genomes do not integrate into the host cell genome but do persist in the nucleus, where genes can be expressed.
• This lack of permanent integration is a benefit in terms of safety, with no danger of insertional mutagenesis or proto-oncogene activation. However, since the viral genome does not replicate, cell division will result in a progressive dilution and eventual loss of the viral DNA.
• Adenoviral treatments are therefore transient or temporary and need to be repeatedly administered to maintain the effect. Wild-type adenoviruses can elicit a powerful immune response, which can be dangerous to the host.
• This led to the first gene therapy death which we will discuss in the next session. Modern strains of therapeutic adenoviruses are engineered to prevent this immune response.

Question 19

What are adeno-associated viruses (AAVs)?

Answer 19

•Adeno-associated viruses (AAVs)

–Discovered whilst isolating adenoviruses

–Do not cause human diseases

–Does not elicit an immune response

–+/-ssDNA genome 4.7 kb

–Usually do not integrate

•Rarely integrate into AAVS1 for latency

Question 20

When were Adeno-associated viruses discovered?

Answer 20

• whilst researchers were trying to isolate adenoviruses from human tissues.
• Unlike the adenoviruses, they do not cause diseases in humans, do not elicit any immune response and are therefore intrinsically safe.
• They have a single-stranded DNA genome which is either sense or antisense and is quite small at approximately 4.7 kilobases.
• This means that there is limited capacity for foreign DNA. Like the adenoviruses, they do not usually integrate into the host cell genome and result in a similar transient transduction.
• However, AAVs can integrate into the genome at a locus on chromosome 19 called AAVS1 for latency.
• Disruption of AAVS1 by insertion is not associated with any human conditions and is interestingly regarded as a safe targeting site for other gene therapies.

Question 21

What are Retroviruses (RVs)?

Answer 21

•Retroviruses (RVs)

–Cause serious diseases in humans

–Do not elicit immune responses

–2x +ssRNA genome 7 – 10 kb

–DNA copy of genome integrates ‘randomly’

•Permanent transduction

–Only infect dividing cells (except lentiviruses)

Question 22

What diseases are retroviruses associated with?

Answer 22

• Retroviruses are associated with a variety of serious diseases in humans, such as HIV infection and AIDS or cancer. They have evolved to efficiently evade the immune system, although they are susceptible to inactivation by serum complement, which limits their in vivo applications.
• Their genome consists of two copies of a sense single-stranded RNA genome which is between 7 – 10 kilobases in size, limiting the capacity for foreign RNA. During the normal RV life cycle the RNA genome is reverse transcribed and replicated to form a dsDNA genome copy which is integrated into the host cell genome by a viral integrase.
• This integration was originally considered to be fairly random, but recent studies have discovered that this is not true. We will find out where the integration sites are in the next session.
• Integration leads to a permanent transduction of the cells and only requires a single treatment. Retroviruses have a broad host range, but are limited in that they will only infect dividing cells. The lentiviruses (HIV-1 is an example) are a group of retroviruses that are able to infect non-dividing cells.

Question 23

Show Gene therapy clinical trials by vector

Answer 23

Adenovirus, retrovirus and naked DNA are the most commonly used vectors. Lentivirus use has increased in recent years and forms the basis for several key successful gene therapy trials.

Question 24

Give examples of gene therapies

Answer 24