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Gene Therapy


The viral vector was administered to mice with the same deficiency as human ... The 240bp band is the human gene, and the 454bp is for the mouse gene. ... – PowerPoint PPT presentation

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Title: Gene Therapy

Gene Therapy Jacqui Hawkins and Sam Marzolf
  • For patients with genetic diseases, can we give
    them new, functional copies of their defective
    gene and relieve their illness?

Answer Yes, in theory
Basic Principles
  • (A) The genetic material of a vector (usually a
    virus) is removed and the normal, functional
    gene is inserted into the viral shell, along with
    any genes necessary for infection.
  • (B) The virus containing the new gene is given
    to a patient where it then infects the defective
  • (C) The viral DNA (along with the new gene)
    inserts itself randomly into the host genome,
    where it hopefully makes functional protein
    product thereby curing the disease.

Picture courtesy of http//
  • How to give therapy (orally, intravenously,
  • How many cells need to be infected for the
    phenotype to become normal?
  • How to cross the blood-brain barrier in diseases
    affecting the brain? Viral vectors are too big.
  • How can we target the right cell type(s) and
    only those cells.
  • Massive immune responses to the viral vector are
    often a problem and can lead to the death of the
    patient, how can this be avoided?
  • Will the infected cells live long enough to
    produce progeny that have the functional gene
    copy incorporated into their genome?
  • Ethical concerns Who decides what is a
    disease and who deserves therapy?

Two closely related papers
  • Whats the disease?
  • MPSVII, a lysosomal storage disease, where an
    essential lysosome enzyme (in this case GUSB) is
    missing or deficient resulting in accumulation of
    cellular junk and lysosomal distention. The
    resulting phenotype includes skeletal, cognitive,
    retinal and auditory components, along with other

Whats the vector? Adeno-associated virus
(AAV), a class of small, single-stranded DNA
viruses that can insert their genetic material at
a specific site, was administered intravenously.
This vector contained human GUSB cDNA, and
several components required for infection and
expression. This vector has been shown to
infect multiple tissues in distant tissues. For
this disease (and many others) where there are
many phenotypic effects, this property is
  • Concepts used?
  • Unique phenomena of cross-correction where
    normal cells secrete lysosomal enzymes that can
    then be taken up by the deficient cell to correct
    its phenotype. This means only a small number of
    cells need to receive the gene replacement for a
    large amount of phenotype correction to be

Secreted protein
Normal Cell
Affected Cell
Normal Cell
Corrected Cell
So, how was gene therapy used?
  • The viral vector was administered to mice with
    the same deficiency as human MPSVII patients two
    days after birth, and their phenotype was
    compared to normal and affected mice at all
    levels and developmental stages.

How did the treated mice compare?
1 normal mouse 2 MPSVII mouse 3 MPSVII
mouse treated at 2 days.
How did the treated mice compare?
Cells staining red indciate GUSB activity, blue
cells are uncorrected. Since the vector infects
many distant tissues, correction was observed in
many different cell types.
How did the treated mice compare?
Analysis of tissues for viral cDNA. The 240bp
band is the human gene, and the 454bp is for the
mouse gene. Mismatches between the PCR primers
and the murine sequence lead to less
amplification of the endogenous gene. There is
more variation and less expression many weeks
after the administration of the AAV. In the 2000
paper a higher dose was used which did last
From the 1998 paper
1 year
From the 2000 paper
How did the treated mice compare?
The survival rates of normal, treated and
untreated mice. The mice treated with the AAV had
a nearly normal life expectancy.
What now?
  • This study has shown that gene therapy can be
    used for long-term correction of MPSVII and most
    likely other simple lysosomal storage diseases,
    however there were some hurdles to over come
  • There was no great affect on the brain because of
    the blood-brain barrier. These diseases are have
    debilitating cognitive effects, so treatment of
    the brain is necessary.
  • Some tissues (like the spleen) rid themselves of
    the cDNA fairly rapidly, so it is necessary to
    develop strategies for dealing with this.

Some other options
  • RNAi How would you do this?
  • Liposomes artificial lipid spheres with DNA
  • Human Artificial Chromosomes (HAC)
  • so far it is hard to get such a large structure
    into a cell nucleus.