Lecture 26

Gene therapy

Mending a bad set of genes

A clarification...

Human gene therapy involves delivery of nucleic acid to a somatic cell to correct a debilitating condition, relieve suffering, and extend life. It does not cause a change in the germ line of the individual, so any genetic corrections are not passed to successive generations.

We already use many specific proteins as therapeutic agents - for example recombinant factor IX used to treat hemophiliacs. It would be better if the patient could simply acquire the cDNA sequence for the wild type factor IX gene in some tissue in its body, and produce enough factor IX to stay healthy.

There are many proteins that cannot be simply purified from a recombinant organism and "infused" into a patient. They would never make it to the right target! For example, the cystic fibrosis transmembrane receptor gene (CFTR) is a membrane protein, so inserting that protein into the cell surface of an epithelial cell in the bronchi would be extremely challenging, especially from the outside. It isn't even that easy from the inside! The most common allele of CFTR causing cystic fibrosis is the deltaF508 mutation (a loss of a phenylalanine 508) that causes the CFTR to not track properly through the ER and Golgi apparatus. The receptor is functional, but it isn't delivered to the right place in the cell.


Development of therapeutic agents

1. Research and discovery
2. Preclinical trials - including in vitro and lab animal studies
3. Phase I trials, used to test the safety of the product on 6-10 human volunteer subjects.
4. Phase II trials, used to test the therapeutic agent on a larger number of human volunteers to see if the drug is helpful
5. Phase III trials, a comprehensive analysis on a large number of human volunteers of the safety and efficacy.


Examples of human gene therapy trials that have been conducted
(source: Glick and Pasternack, Molecular Biotechnology, 1998, Table 21.2)
Condition Therapy Target cells
Adenosine deaminase deficiency Adenosine deaminase Lymphocytes, bone marrow cells
Melanoma Tumor necrosis factor Tumor-infiltrating lymphocytes, autologous tumor cells
Melanoma, glioblastoma, renal cell cancer IL-2 Autologous tumor cells
Hemophilia B Factor IX Autologous skin fibroblasts
Hypercholesterolemia LDL receptor Autologous liver cells
Melanoma, colorectal cancer, renal cell cancer Histocompatibility locus antigen class I-B7 plus beta 2 microglobulin Tumor cells
Glioblastoma, AIDS, ovarian cancer HSV-TK Tumor cells, T-cells
Cystic fibrosis Cystic fibrosis transmembrane receptor Nasal and airway epithelia
Breast cancer Multidrug resistance Blood CD34+ cells
Melanoma GMCSF Tumor cells
Arthritis IL-1 receptor agonist Autologous fibroblasts
Amyotrophic lateral sclerosis Ciliary neurotrophic factor Encaplulated transduced xenogeneic cells
Head and neck squamous carcinoma p53 Tumor cells
Fanconi anemia Fanconi anemia C Bone marrow cells

Here are some things you'll want to think about.

If you want to develop a cure for a disease by gene therapy, here are some things to think about:

  • How will you reach the target cells and deliver the gene?
    • ex vivo vs. in vivo
  • What proportion of target cells need to be altered?
  • Does the gene need to be expressed constitutively, or regulated?
  • Would there be serious consequences if the gene were overexpressed?
  • How long will the DNA persist and be expressed?
  • If you are planning to modulate gene expression, how will you do it (e.g. ribozymes, antisense, short interfering RNAs - siRNA)?

Delivering the gene by retroviral vector

Here is the structure of a typical retrovirus genome:

The salient features include 5' and 3' LTRs (long terminal repeats), a gag (group specific antigen gene, or internal capside protein gene), pol (reverse transcriptase), and env (envelope protein). A critical element for packaging is the psi sequence, shown in red, which is a cis-acting element. That is, an RNA must have a psi sequence if it is to be packaged. The protein coding genes are trans acting, of course, because they may diffuse around the cytoplasm.

A modified retrovirus for gene therapy might look something like this, with psi sequence, a gene of interest at a multiple cloning site, and neomycin resistance gene (driven from its own promoter, or using an IRES element for internal ribosome entry as in the example below).


This would need to be made in a "helper cell" that expressed gag, pol, and env in trans from genomic DNA.

For example:
Clontech's Retroviral Packaging Cell Line - AmphoPack˘-293 Cell Line

"The AmphoPackTM-293 Cell Line can be used to produce viral particles that infect a broad range of mammalian cells. AmphoPack-293 is derived from a human embryonic kidney cell line (HEK 293) that is easily transfected, and produces virus in titers that can exceed 106 cfu/ml. The viral envelope protein expressed by AmphoPack-293 recognizes the amphotropic receptor, allowing foreign genes to be delivered to a range of mammalian cells including mouse, rat, human, hamster, mink, cat, dog, and monkey cell lines. AmphoPack-293 Cells can produce high-titer virus 4872 hours after transfection" http://www.clontech.com/products/catalog02/HTML/1069.shtml

To make sure that no viral particles containing the gag, pol, and env genes were created, the trans acting genes could be dispersed to several loci.

They explain further:

"Integrated in to the packaging cell line genome are the gag, pol, and env genes necessary for viral reproduction. The retroviral vector provides the RNA packaging signal, transcription and processing elements, and target gene. The packaged viral particles acquire envelope glycoproteins from the packaging cell's membrane as they bud from the cell. These proteins determine the type of receptors the virus uses to infect host cells. The viral particles produced by the packaging cell lines are replication-incompetent since they do not carry the genes necessary for viral reproduction." http://www.clontech.com/retroviral/index.shtml

Now I can hear you thinking ... Right! Does that really work, and what happens if a few replication competent retroviruses (RCR) slip through? Well - that is a problem, and the FDA has established
guidance for the industry for how to test samples for RCR. As noted in the report:

"The overriding safety issues associated with the use of retroviral vectors are exemplified by the findings of an experiment involving administration of ex vivo transduced bone marrow progenitor cells that had been inadvertently exposed to high titer RCR contained in the retroviral vector material to severely immunosuppressed Rhesus monkeys. In this setting, 3/10 animals developed lymphomas and died within 200 days" http://www.fda.gov/cber/gdlns/retrogt1000.htm#ii

Now I can hear you thinking...O.K., I'll buy that, but how does this actually work? The retrovirus injects an RNA copy of the recombinant sequence, and it doesn't inject a pol (reverse transcriptase) gene? How are you going to keep this in a cell as DNA? Don't worry - the viral particle includes the reverse transcriptase enzyme as a protein, which is used to make the cDNA of the viral genome. The cDNA integrates into the genome and is preserved.

Resource reading

Retroviral vectors - David Peel. Univ. of Leicester

Gene interference

A word or two about gene modulation with siRNA.

Short interfering RNAs are 21-23 bp double stranded RNAs that can initiate the enzymatic breakdown of specific mRNAs in a cell, through an RNA induced silencing complex. Here's a figure from MWG Biotech, explaining the process:


An example of an application is explained by Invitrogen Inc., and they show the "knockdown" of a specific transcript Lamin A/C without loss of a nonspecific one.

"HeLa cells were plated at 6 X104 cells per single well of a 24 well plate in DMEM with 10% FBS 24 hours before transfection, resulting in 90% confluence the day of transfection. 60pMol of siRNA Lamin A/C (Dharmacon) or GFP siRNAs (Xeragon). siRNAs were resuspended following the manufactures protocols and diluted in 50 micro-l of Opti-MEM(R) in a separate tube 2 micro-l of Lipofectamine(TM) 2000 was diluted in 48 µl Opti-MEM(R). The diluted siRNAs and diluted Lipofectamine˘ 2000 were then combined, gently mixed and allowed to incubate for 20 minutes at room temperature. The siRNA:Lipofectamine˘ 2000 mixture was added directly to the cells. The media was replaced with fresh media 4 hours after transfection. Western blots were generated two days following transfection. The cells were removed from the plate and lysed with 40 micro-l of LDS loading buffer and run on a 4-12% Tris-Bis NuPAGE gel. The samples were then transferred to PVDF membrane and blotted with anti-Lamin A/C antibodies (Santa Cruz Biotech) (panel A). Following detection of Lamin A/C the blots were stripped and reprobed with anti-actin antibodies. Detection was accomplished using the Western Breeze kit (panel B)." http://www.invitrogen.com/content.cfm?pageid=4547

Here is a histogram from Invitrogen showing several different cell line results:


Resource reading

User guide - Tuschl lab

Pseudotyping and cell targeting

Here are some tricks that may seem especially important if you are planning in vivo, rather than ex vivo gene therapy. In ex vivo therapy you are removing cells from a patient (autologous cells), treating them, and returning them to the patient as a graft. In in vivo therapy, you treat a person directly with the viral vector and would a specific cell type to be targeted.

Envelope proteins of retroviruses are specific for viral receptors on cell surfaces, and so this is the key to specificity of infection. You may change the envelope protein expressed on a virus to change the "host range" or type of cell infected, and this is called
pseudotyping. For example, retroviral packaging cell lines may be "ecotropic" (for infection of mouse or rat cells), "dualtropic", "amphitropic" (for infection of a wider variety of mammalian cells", or "pantropic" (for a very wide host range including some non-mammalian cells). The more refined version of this, provided to us by genetic engineering, is to change the env gene so that it encodes a short peptide sequence that will bind to a target cell.

Suppose, just for example, that you wanted to infect HIV-positive cells with a recombinant retrovirus expressing the thymidine kinase gene from HIV (so that you could kill them specifically with ganciclovir). How could you target HIV infected cells? Well, HIV expresses its own envelope proteins and these go to the cell surface during expression so that the viral capsid can pick them up during viral budding.

You could engineer into the therapeutic virus env gene, a peptide that binds to the env protein of the HIV. What might that be? Why, CXCR-4 of course!

When the HIV-infected cell is infected with this recombinant retrovirus (by virtue of the association of the HIV-env protein with the segment of CXCR-4 fused into the recombinant), the TK gene is expressed in the infected cell. Upon treatment with ganciclovir, it's all over for the cell!

Adenovirus-mediated gene therapy

Retroviruses are not the only types of viruses being used for in vivo or ex vivo gene therapy.

Adenovirus is a double-stranded DNA virus that is particularly good at infecting epithelial cells. In nature, adenoviruses are one of the causes of colds and respiratory infections.

The E1 genes of adenovirus are required for lytic growth, so you can make a recombinant adenovirus that is replication defective by integrating a gene of interest (a therapeutic gene, let us say) into the E1 locus:

It would be best to start with a version of adenovirus in which the E1 gene was already deleted and one that could not be packaged, so as not to generate progeny virus that were replication competent. The recombination event is designed to make the genome packageable -- now all we have to worry about is E1.

The recombination takes place in a cell that expresses the E1 genes in trans from a chromosomal locus, so the recombinant genome can be packaged.

There have been some difficulties with adenovirus vectors, in part because there is some leaky expression of the genes even in the absence of E1 (i.e. in a transduced cell).

Quotations from an article regarding a gene therapy trial tragedy:

Science. Volume 286, Number 5448, Issue of 17 Dec 1999, pp. 2244-2245. Gene Therapy Death Prompts Review of Adenovirus Vector. By Eliot Marshall

"For the past 3 months, faculty and staff members at the University of Pennsylvania's Institute for Human Gene Therapy have been trying to understand why a relatively fit 18-year-old with an inherited enzyme deficiency died on 17 September, 4 days after doctors at Penn injected a genetically altered virus into his liver."...

"Gelsinger was the first patient in a gene therapy trial to die of the therapy itself, as James Wilson, who heads the Penn institute, confirmed at a public meeting..."

"Wilson, the chief of Penn's clinical team, appeared with co-investigators Mark Batshaw and Steven Raper at a special public meeting at the National Institutes of Health (NIH) in Bethesda, Maryland, on 8 and 9 December to examine what went wrong. ... After releasing stacks of clinical data and answering questions for 2 days, however, Wilson and colleagues said that they didn't fully understand what had gone amiss. They reported that the vector they used--a crippled form of adenovirus combined with a gene to control Gelsinger's ammonia metabolism (the gene for ornithine-transcarbamylase, or OTC)--invaded not just the intended target, the liver, but many other organs..."

"This triggered an "activation of innate immunity," the Penn clinicians wrote, followed by a "systemic inflammatory response." Within hours, Gelsinger's temperature shot up to 104.5 degrees Fahrenheit. He went into a coma on the second day and was put on dialysis and then on a ventilator. His lungs filled with fluid. When it became impossible to oxygenate his blood adequately, he died."

"The Penn team had given Gelsinger a massive dose of the vector--38 trillion virus particles, the highest dose in this 18-patient trial--to try to get enough functioning OTC genes into his liver. But even so, only 1% of the transferred genes reached the target cells."

Stratagene Inc. has an AdEasy vector system for adenovirus transduction, and they write:

"Adenoviruses are capable of infecting a broad range of cell types and infrection is not dependent on active host cell division. Protein production techniques in mammalian cells require high titers and high-level gene expression, both of which can be achieved using adenoviral vectors. Adenoviral vectors can be used to overexpress recombinant proteins in mammalian cells. As a result, the resulting proteins have the relevant posttranslational modifications and folding, which is not possible when overexpressing proteins in prokaryotic systems."

Resource reading:

Adenoviral vectors - David Peel. Univ. of Leicester
Viral Vectors and Gene Therapy - Planelles. Univ. Rochester
Report from the Gene Therapy meeting 4th July 2002

More on the problem of stability in vector systems

FDA survey

Adeno-associated virus

Adeno associated virus (AAV) is a parvovirus that has been used as a vector to transfer DNA to cells.

For example, Stratagene Inc. has a wide variety of recombinant AAV vectors


The pCMV-MCS is the shuttle vector, and it is co-transfected into a packaging cell line with the pAAV-RC and pHelper vector.

The packaging cell line produces helper-free recombinant AAV which can be used for the transduction of target cells. Here is their flow chart:

Stratagene writes:

"AAV is naturally replication-deficient and normally requires coinfection with a unrelated helper virus, like adenovirus, to generate AAV virions. This novel system uses a vector containing the necessary genes from adenovirus (pHelper vector) to induce the lytic phase of AAV producing recombinant, replication-defective AAV virions ready to deliver a gene of interest to target cells."

"Recombinant adeno-associated virus (AAV) is a proven research and therapeutic tool. This system is used to introduce genes into cells for gene expression or gene therapy studies. Using this system, genes can be delivered into a wide range of hosts including many different human and non-human cell lines or tissues."



Herpesvirus methods

Here's an interesting idea. You can package a gene of interest in a herpesvirus capsid (e.g. herpesvirus saimiri) by generating a cycle of lytic replication, and packaging a rolling-circle multimer as a "head full" for the virus.

Epstein-Barr Virus can also be used as a cloning vector, and can be stably maintained in cells by virtue of an origin of replication (oriP) used in latent infection. For example:

"The UNCCH laboratory has developed the HAEC system to establish large DNA fragments as episomes in human cells, using the latent replication elements from the human herpes Epstein-Barr virus (EBV). A first-generation episomal vector based on the latent origin of replication oriP and its transactivator EBNA-1 from EBV was used to establish and maintain up to 350 kb of circular DNA in human cells. Such a system allowed the generation of a random clone library covering 10% to 20% of the human genome as extrachromosomal self-replicating episomes in human cells." http://www.ornl.gov/hgmis/publicat/hgn/v9n1/haec.html