Viral Vectors and Gene Therapy

Question 1

What is gene therapy?

Answer 1

a novel approach to treating diseases based on modifying the expression of a person’s genes toward a therapeutic goal

Take home message #1: Gene therapy remains a highly experimental collection of technologies whose full potential is yet to be realized.

Question 2

Somatic gene therapy

Answer 2

Involves the manipulation of gene expression in cells so as to be corrective for the patient, but this correction is not inherited by the next generation

Question 3

Germline gene therapy

Answer 3

Involves the genetic modification of germ cells that will pass the selected change on to the next generation.

germline intervention is strictly limited to animal models and there is no intent to pursue this type of approach in humans currently

Question 4

How is ex vivo, somatic cell gene therapy performed?

Answer 4

1. A piece of the patient’s liver was removed, and the cells were treated with a retrovirus carrying the low- density lipoprotein (LDL) receptor gene.
2. Liver cells failing to take up corrective genes were discarded; cells incorporating the corrective gene were reimplanted into the patients’ liver.

Question 5

What are the the main 4 indications that are candidates for gene therapy?

Answer 5

1) Cancer;
2) Genetic deficiencies caused by single gene mutations (e.g. monogenic diseases, OTC deficiency, LPL deficiency, PKU, Hemophilia A and B, Sickle cell anemia, etc.);
3) Infectious diseases (HIV); and
4) Cardiovascular diseases.


Question 6

2nd take-home message:

Answer 6

There are no FDA approved gene therapy products currently on the market (in the U.S.). All gene therapy interventions are in various stages of clinical trials or in the preclinical stage; however, the first commercial gene therapy product (Glybera) was just approved in November, 2012 in Europe for lipoprotein lipase deficiency (LPLD).

Question 7

Two types of intervention strategies for gene therapy applications:

Answer 7

Therapeutic and Cytolytic Strategies

Question 8

What is the basis of therapeutic strategies for gene therapy?

Answer 8

Vector carries a gene that encodes a protein that is either defective or that is not present due to mutation(s) in the patients’ endogenous gene(s).

Question 9

What is the basis of cytolytic strategies for gene therapy?

Answer 9

Vector is designed to destroy or eliminate a diseased cell or tissue. Example shows a virus carrying the gene for thymidine kinase (TK) from herpes simplex virus. Expression of TK converts the prodrug gancyclovir to the toxic product gancyclovir phosphate.

Many gene therapies applications for cancer involve cytolytic strategies.


Question 10

What are vectors?

Answer 10

Vectors are generally modified, attenuated viruses that are used to deliver a payload (gene) into a cell.

Question 11

Requirement and Challenges – Steps in the development of a genetic therapy

Answer 11

A. Gene identification and cloning
–Must have identified the gene (or genes) responsible for a particular disease state and have demonstrated concordance of disease state with a defect in the particular gene or gene product. –This has become much easier with the completion of the Human Genome Project couple with “’omics” analysis to define gene interaction networks.

B. Delivery
–First task in any gene therapy strategy is delivery of genetic material to the appropriate cells of the
patient in a way that is specific, efficient and safe.
1. Involves the development of gene delivery vehicles (vectors)
a) Most vectors are based on attenuated or modified versions of viruses.
–Challenge is to remove the disease-causing components of the virus and insert recombinant genes that will be therapeutic to the patient.
b) Another strategy utilizes non-viral vectors
–Liposome transfection - Complexes of DNA and lipids
–“Gene gun” technology - Delivery of DNA on gold particles (primarily for DNA vaccines)

C. Elements to ensure controlled gene expression

• -Provides means to make the correct amount of therapeutic protein at the right time.
• -Essential for maintaining long-term expression of the gene in the correct cells or tissue.

D. Understanding of the host immune response

• -Important to use of vectors that give minimal or no adverse immune responses
• -Essential if there is a need to give multiple doses of the vector (such as with adenovirus vectors or any vector that does not integrate into the host genome).

Question 12

What are some types of vectors?

Answer 12

Adenovirus
Adeno-associated virus
Herpesvirus
Liposomes/Naked DNA
Retrovirus

Question 13

What are the properties of Adenovirus?

Answer 13

• Episomal
• High transduction efficiency
• Infects replicating and non-replicating cells - Elicits an immune response
• Insert capacity 8-36kb

A5- GEI836 (➗+)

Question 14

What are the properties of Adeno-associated virus?

Answer 14

• Integrates genome into specific region on human chromosome 19 - Low immunogenicity (Does not elicit antibodies against itself)
• Does not stimulate inflammation in the host)
• No associated disease
• Infects both dividing and non-dividing cells
• Limited insert capacity of ~5kb

AA6- H19(IID-)5 (➗+)

Question 15

What are the properties of Herpesvirus?

Answer 15

• Large insert capacity
• Broad host range
• Infects dividing and non-dividing cells

H3-LIC/BHR (➗+)

Question 16

What are the properties of Liposomes/Naked DNA?

Answer 16

• No limit to the size of genes that can be delivered
• Low immunogenicity
• Poor levels of gene transfer

L3- (IG-), SIZE+

Question 17

What are the properties of Retrovirus?

Answer 17

• Non-pathogenic in humans
• Stably transduces dividing but not non-dividing cells
• Inserts genome into host cell’s DNA
• Long term expression
• Insert capacity of 8kb
• Inactivated by human complement

R6- H(I^c)8 (D➗-), HLTE

Question 18

What are some major problems that physicians need to address before effective use of vectors?

Answer 18

A. How to avoid an immune response in the patient.
–Vector components can provoke inflammation (a serious problem with adenovirus vectors)

B. How to get genes into non-dividing cells like liver, muscle, and neurons.

• -This is a problem that is dependent on the type of vector used. For example, MLV-based retrovirus vector require dividing cells to get the viral genome into the host nucleus.
• HIV (Lentivirus) vectors do not have this restriction and is one of the advantages of these vectors.

–Specific targeting of particular cells types still remains a significant problem. Most vectors do not infect a specific target cell, but rather target those cells that the vector normally infects.

C. How to get the gene integrated so that it will be replicated and expressed indefinitely and as needed.

Question 19

What are retroviruses?

Answer 19

Enveloped viruses that have an RNA genome that, upon infection of a cell, is converted to dsDNA by the enzyme reverse transcriptase. For infection, virus binds to a specific receptor on the host cell membrane and after uncoating, the RNA genome is reverse transcribed and then the dsDNA genome is delivered into the cell nucleus where it integrates and therefore becomes a part of the host cell genome. This ability to integrate (in theory) allows for long-term expression and allows the therapeutic gene to be maintained during cell division

Question 20

How can retroviruses be used in gene therapy?

Answer 20

To generate a gene therapy vector with retroviruses the viral genes encoding the structural proteins (receptor binding protein and capsid proteins) are deleted and replaced with the therapeutic gene of interest.


Question 21

What is one of the disadvantages of murine-based retrovirus vectors?

Answer 21

they require replicating cells for genome integration, which limits their use.

Lentiviruses (or HIV-based gene therapy vectors, which are a sub-type of retrovirus) can integrate into both replicating and non-replicating cells, so most gene therapy vectors used today are based on lentivirus vectors.


Question 22

What are adenoviruses?

Answer 22

Non-enveloped viruses with a large dsDNA genome that can accommodate a large gene inserts (up to ~36k bp). There are over 51 different serotypes of adenoviruses in humans and adenoviruses are responsible for 5–10% of upper respiratory infections in children and adults.

Hence all of us have seen adenoviruses and have an immunological memory to them.

Question 23

How do adenoviruses enter cells?

Answer 23

Adenovirus enters cells by binding to specific receptors and following virus uncoating the genome is delivered into the nucleus, but in contrast to retroviruses, adenovirus genomes do not integrate, but instead replicate episomally.

Like retrovirus vectors, adeno-vectors have the genes for the structural proteins and proteins that induce host cell division (such as the E1A gene product) deleted. Sometime these are called “gut-less” vectors because they lack almost all viral genes and carry only the therapeutic gene of interest.


Question 24

What are Liposomes?

Answer 24

formulations of synthetic lipids that bind to and encapsulate plasmid DNA which then fuse with cell membranes to deliver the genetic payload into cells. Liposomes do not bind to specific cell receptors and therefore can deliver therapeutic genes into many different cell types, but generally the efficiency is much lower than for viral vectors.


Question 25

How are gene therapy vectors made?

Answer 25

gene therapy vectors are constructed from viral genomes in which the structural proteins are removed. This eliminates the possibility that the vector can become a self-replicating virus that can spread on it’s own.

Question 26

How are the structural proteins of gene therapy vectors removed?

Answer 26

First, the genes encoding the structural proteins are deleted from the genome and cloned into a plasmid expression vector that is then transfected into a cell such that the cell can express the structural proteins, but no viral genomes. This is done in cell culture and the cell is then called a “packaging cell line”.
 To the produce a gene therapy vector, sequences from the virus that result in packaging of the genetic material into virus particles (called the packaging signals) are removed, cloned into a plasmid, and the therapeutic gene of interest is cloned between the packaging signals.
 When this plasmid with the packaging signals and therapeutic gene is introduced into the packaging cell line, the structural proteins assemble the therapeutic gene into virus particles, which are then used as the gene therapy vehicle (or vector). This is now called the “producer cell line”. 
The virus particles (or vectors) that are released from the packaging cell can infect new cells (such as those removed from patients in an ex vivo strategy, or if given directly to a patient in an in vivo strategy), but because the genome lacks the genes encoding the structural proteins, they only deliver the therapeutic gene into cells, but no other infectious viruses are made since those cells (in the patient) do not encode structural proteins of the virus.


Question 27

What are adeno-associated viruses (AAV)?

Answer 27

a small virus which infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response, which is a major advantage for a gene therapy vector. 
 AAV belongs to the parvovirus family, which are non-enveloped viruses that have ssDNA genomes.

Question 28

What is Glybera?

Answer 28

The first commercial gene therapy vector that received approval in Europe (Glybera) is an AAV vector carrying the lipoprotein lipase gene. Patient’s with LPL deficiency cannot breakdown fats. This is a rare disease affecting only 1 out of 1 million. Symptoms of familial LPL deficiency usually begin in childhood and include abdominal pain, acute and recurrent pancreatitis, eruptive cutaneous xanthoma and hepatosplenomegaly.


Question 29

What is SCID-X1?

Answer 29

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation.

Question 30

What is SCID-X1 caused by?

Answer 30

This block is caused by mutations of the gene encoding the gamma cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors.

Question 31

Gene Therapy for SCID-X1?

Answer 31

After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gamma Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells (hematopoietic stem cells). After a 10-month follow-up period, gamma transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit. The gene transfer approach of the Paris clinical trial has led to life-saving immune recovery in 10/11 (approximately 90%) of the treated infants, who were able to go home.

Question 32

Correction of ADA-SCID by Stem Cell Gene Therapy Combined with Nonmyeloablative Conditioning. Aiuti, A. et al. Science (2002) 296:2410-2413.

Answer 32

Purine metabolic defect which causes impaired lymphocyte development and function Trial involved two patients: Pt. 1- diagnoses at birth and was a sibling of two other children with ADA-SCID. Had recurrent pulmonary infections before therapy Pt. 2- had a sibling who died at 3.5 months of an undefined primary immunodeficiency. Suffered from pneumonia, diarrhea, and scabies and ADA-SCID. Therapy used a retrovirus-derived vector containing the ADA cDNA and ex vivo infection of CD34+ cells Hematopoietic stem cell (HSC) gene therapy for adenosine deaminase (ADA)-deficient severe combined immunodeficiency (SCID) has shown limited clinical efficacy because of the small proportion of engrafted genetically corrected HSCs. We describe an improved protocol for gene transfer into HSCs associated with nonmyeloablative conditioning. This protocol was used in two patients for whom enzyme replacement therapy was not available, which allowed the effect of gene therapy alone to be evaluated. Sustained engraftment of engineered HSCs with differentiation into multiple lineages resulted in increased lymphocyte counts, improved immune functions (including antigen-specific responses), and lower toxic metabolites. Both patients are currently at home and clinically well, with normal growth and development. These results indicate the safety and efficacy of HSC gene therapy combined with nonmyeloablative conditioning for the treatment of SCID.

Question 33

Treatment of leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: short-term results of a phase I trial. Hauswirth et al., Hum Gene Ther. (2008) 19:979-90.

Answer 33

Leber congenital amaurosis (LCA) is a group of autosomal recessive blinding retinal diseases that are incurable. One molecular form is caused by mutations in the RPE65 (retinal pigment epithelium-specific 65-kDa) gene. A recombinant adeno-associated virus serotype 2 (rAAV2) vector, altered to carry the human RPE65 gene (rAAV2-CBSB-hRPE65), restored vision in animal models with RPE65 deficiency. A clinical trial was designed to assess the safety of rAAV2-CBSB- hRPE65 in subjects with RPE65-LCA. Three young adults (ages 21-24 years) with RPE65-LCA received a uniocular subretinal injection of 5.96 x 10(10) vector genomes in 150 microl and were studied with follow-up examinations for 90 days. Ocular safety, the primary outcome, was assessed by clinical eye examination. Visual function was measured by visual acuity and dark-adapted full-field sensitivity testing (FST); central retinal structure was monitored by optical coherence tomography (OCT). Neither vector-related serious adverse events nor systemic toxicities were detected. Visual acuity was not significantly different from baseline; one patient showed retinal thinning at the fovea by OCT. All patients self-reported increased visual sensitivity in the study eye compared with their control eye, especially noticeable under reduced ambient light conditions. The dark-adapted FST results were compared between baseline and 30-90 days after treatment. For study eyes, sensitivity increases from mean baseline were highly significant (p

Question 34

SETBACKS | A. Death of the first patient in a gene therapy trial

Answer 34

Jesse Gelsinger was an Arizona teenager who died on September 17, 1999 soon after receiving adenovirus-based gene therapy for treatment of partial ornithine transcarbamylase (OTC) deficiency—an X-linked defect of the urea cycle in which nitrogen metabolism is affected, leading to a spectrum of neurological symptoms including seizures and mental retardation. Therapy for the condition relies on alternative substrate administration, but mortality rates with the disease are high. Gelsinger's death is the first mortality directly attributed to gene therapy in a total of about 5,000 patients treated since the first gene therapy trial was conducted by Dr. W. French Anderson in 1990. The morning immediately after administration of the gene therapy vector Gelsinger’s condition had deteriorated so seriously that he was placed in intensive care. The alternate pathway therapy failed, and by the second evening he was comatose. Doctors improved his condition temporarily, but he developed acute respiratory distress syndrome (ARDS) and died two days later of multiple organ failure due to anoxia. Measurements of inflammatory cytokines suggested that the vector had caused systemic inflammatory response syndrome (SIRS), which is associated with ARDS. The following are some of the details about Gelsinger's death. First, the vector was found throughout his body, in especially high concentrations in the spleen, bone marrow, lymphatic tissue and gonads. Second, his bone marrow was completely depleted of precursor cells, which some believe could not have occurred in the few days following vector infusion. There was evidence of parvovirus, of unknown origin, which the lead scientist believes could have caused the bone marrow to become depleted and possibly to have pushed Gelsinger's immune system into overdrive. In addition, unlike other patients given the vector, Gelsinger's IL-6 levels rose but never declined, and his IL-10 levels never rose sufficiently. This might have been a result of his fever, parvo infection, or both.

Question 35

Serious Adverse Event in a Clinical Trial of Gene Therapy for the X-Linked Form of Severe Combined Immune Deficiency Disease (X-SCID) January 15, 2003:

Answer 35

B. U.S. officials suspend 27 more gene therapy studies while they investigate a possible serious risk: A second toddler cured of the deadly "bubble boy disease" by gene therapy in France has come down with an apparent leukemia-like side effect. Two serious adverse events were observed in the clinical trial of gene therapy for the X-linked form of severe combined immune deficiency disease (XSCID) being performed by Alain Fischer, MD and colleagues at the Hôpital Necker Enfants Malade, Paris, France. In October, 2002 one of the subjects developed what appears to be T cell leukemia-like illness, about three years after the gene therapy procedure. The child is now receiving chemotherapy. A second child was diagnosed with a similar leukemia in December 2002. The cause of this leukemia appears to be consequence of a process termed "insertional oncogenesis." Insertional oncogenesis has been previously thought to be a very unlikely but possible risk with the type of vector used in this study, since these vectors cannot reproduce themselves and so cannot repeatedly insert into the cell's chromosomes, the process that is most likely to lead to a malignant change. No evidence of leukemias or other forms of cancer were seen in the extensive pre-clinical studies performed before the trial was begun. Although no similar adverse event has been thought to be related to the gene therapy in more than 100 patients, the success rate and detail of follow-up has been much more extensive in the Fischer trial. This risk of cancer as an adverse side-effect of gene therapy is stated explicitly in the informed consent statements that describe the risks and potential benefits to patients and their families.

Question 36

Take home message #3: As with all clinical research, patient safety is of paramount importance and all possible efforts must be made to minimize risks.

Answer 36

Take home message #3: As with all clinical research, patient safety is of paramount importance and all possible efforts must be made to minimize risks.

Question 37

Factors that will influence the development of gene therapy in the future include:

Answer 37

A. Optimizing vector safety to reduce adverse event risk B. Overcoming the technological obstacles C. Additional analysis of the human genome, including understanding of microRNAs D. Expansion of the diagnostic industry E. Reducing risk to patients which is essential for public acceptance of gene therapy

Question 38

CRISPR/Cas

Answer 38

A revolutionary new development for gene therapy applications The CRISPR-Cas9 system allows for specific gene editing (silencing, enhancing or exchanging genes). By expressing Cas9 and specifically designed CRISPRs, any organism's genome can be cut at any desired location allowing specific replacement of a defective gene with a functional gene.