|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
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,
|Adenosine deaminase deficiency
||Lymphocytes, bone marrow cells
||Tumor necrosis factor
||Tumor-infiltrating lymphocytes, autologous tumor cells
|Melanoma, glioblastoma, renal cell cancer
||Autologous tumor cells
||Autologous skin fibroblasts
||Autologous liver cells
|Melanoma, colorectal cancer, renal cell cancer
||Histocompatibility locus antigen class I-B7 plus beta 2 microglobulin
|Glioblastoma, AIDS, ovarian cancer
||Tumor cells, T-cells
||Cystic fibrosis transmembrane receptor
||Nasal and airway epithelia
||Blood CD34+ cells
||IL-1 receptor agonist
|Amyotrophic lateral sclerosis
||Ciliary neurotrophic factor
||Encaplulated transduced xenogeneic cells
|Head and neck squamous carcinoma
||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?
- 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
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.
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.
vectors - David Peel. Univ. of Leicester
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:
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
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
|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
"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."
vectors - David Peel. Univ. of Leicester
Viral Vectors and Gene Therapy - Planelles. Univ.
Report from the Gene Therapy meeting 4th July 2002
More on the problem of stability in vector systems
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:
"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."
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
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
"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."