Dec
2018
29
F
Feature
making heritable changes to the human
embryonic genome remains prohibited
under the Prohibition of Human Cloning
for Reproduction Act 2002, although some
research may be authorised under the Research
Involving Human Embryos Act 2002.
Nevertheless, a picture is starting to
emerge of the areas in which CRISPR may
have the most therapeutic impact.
“Correcting a specific gene defect that’s
known to result in a disease phenotype gives
you potentially a permanent and highly
effective solution to the disorder,” says
Professor Koopman, who is from the division
of genomics of development and disease at
UQ’s Institute for Molecular Bioscience.
“It’s not prohibitively expensive compared
to some types of drug therapy and it’s
fairly quick.”
Broadly, CRISPR can be applied to two
types of therapy — germline gene therapy and
somatic gene therapy.
Germline gene therapy attracts the charge
of ‘playing God’ by making a permanent,
heritable change to a person’s DNA to correct
a genetic mutation.
“It comes with attendant risk that you
might not only be fixing the mutation you
want to correct, but you might also be
introducing unforeseen and undesirable
mutations to subsequent generations,”
Professor Koopman says.
“This is a deeply societal ethical issue.
We have enough controversy genetically
modifying crops let alone the human race. But
critics focus on the potential risk and dangers,
rather than the actual risk and dangers.”
One of his areas of interest is differences
of sex development. When a lab communicates
a candidate gene mutation for causing a
sex development difference in a patient,
his team uses CRISPR to create a mouse
copy of that mutation.
“If that mimics the human phenotype, we
can say with greater certainty that the specific
gene and that mutation is causal in human
differences of sex development. We can feed
that into the diagnostic pipeline for disorders
of sex development,” he says.
The second type — somatic gene therapy,
in which a patient with a gene disorder can be
treated by correcting a group of the patient’s
cells or a whole organ — is not as contentious.
This type of gene therapy comes with
greater technical challenges, such as the
effective redelivery of sufficient corrected cells
back to the patient.
However, some loss of function disorders
result from having too little of a particular
gene product. If, for example, a small number
of functional CFTR cells were introduced
into a patient with cystic fibrosis, then some
lung function could potentially be restored,
according to Professor Koopman.
Some of the pioneering work to come
out of Australia involves using CRISPR
to introduce beneficial natural mutations
into blood cells to boost their production
of fetal haemoglobin. It has been viewed as
a forerunner to gene therapy for a range of
common inherited blood disorders, including
sickle cell anaemia and beta thalassaemia.
A UNSW team found that two genes,
BCL11A and ZBTB7A, switch off the fetal
haemoglobin gene by binding directly to it.
But, in some patients with reduced symptoms,
beneficial mutations work by disrupting
the two sites where these two genes bind,
effectively switching the gene back on.
“I think we will see a handful of big wins
in treatment of blood diseases, rare genetic
eye diseases and cancer via immunotherapy,”
says Professor Merlin Crossley, a molecular
biologist at UNSW Sydney.
“But the main gain will relate to across-
the-board advances in research. We have
knocked down a wall because we can
manipulate genes in cell cultures, which
greatly improves our ability to find knowledge
about almost any biological system.”
This approach is being practised at the
Gene Editing Core Facility at the Murdoch
Children’s Research Institute, Melbourne,
where kidney tissue is created from stem cells,
and genes involved in kidney development
are ‘tagged’.
“Skin cells or blood cells are collected from
kidney patients with a known mutation in a
gene that’s involved in kidney disease,” says
the facility’s director, Dr Sara Howden.
“We then use gene editing to correct that
mutation and make ‘little kidneys’ from stem
cells that have and haven’t been corrected, and
compare them to try and understand why the
mutations cause kidney disease.
“Pharmaceutical companies are
particularly interested in using kidney
tissue for drug screening because a lot of
their drugs will fail phase I trials because
of kidney toxicity.”
Elsewhere, scientists in Japan have recently
used CRISPR to successfully block the HIV
virus from replicating inside infected cells by
removing two regulatory genes of HIV-1 that
cause 95% of HIV infections, and so stop
further production of the virus. 1
“Based on the rapid advances being
achieved in CRISPR/Cas9 research, a HIV-1
functional cure may soon be within reach,”
the researchers claimed, adding that there
were no off-target effects seen from the
genome editing, and it did not affect the
How it works
CRISPR/Cas9 technology has been adapted
from nature. Bacteria use the RNA-guided
enzyme Cas9 like a pair of molecular
scissors to cut up the DNA of invading
viruses that might otherwise kill them.
This mechanism has been adapted by
scientists to edit an organism’s DNA code to
correct a genetic mutation, or to enhance the
genetic code of crops, livestock and humans.
CRISPR (Clustered Regularly Interspaced
Short Palindromic Repeats) refers to the
repeat DNA sequences that were part of a
complex system telling the ‘scissors’ which
part of the DNA to cut.
The technology enables a targeted
segment of DNA to be cut from a cell or
for the entire genome to be edited. The cell
repairs the break using the available DNA
from a replacement gene, which has been
injected or delivered by other techniques.
Alternatively, if no new DNA is available,
the cell will repair the cut by joining the
ends, often disrupting the function of the
gene because some of the DNA is lost
at the cut site.
The latest CRISPR genome editing
systems do not require a DNA cut because,
unlike the ‘blunt’ scissors of the original
editing system, the new precision-guided
systems can rewrite individual letters, or
genetic bases, by delivering an enzyme
to a particular gene to alter it.
survival of the cultured cells.
However, two other recent reports
published in Nature Medicine have raised
the spectre of cells with genomes successfully
edited by CRISPR-Cas9 having the potential
to cause tumours inside a patient. 2,3
The injury that CRISPR/Cas9 causes to a
cell activates a ‘first-aid kit’ orchestrated by
the p53 gene. But the edited cells that survive
this repair mechanism have a dysfunctional
p53 and, according to the research, p53
mutations are responsible for many cancers,
including nearly half of ovarian cancers, 43%
of colorectal cancers and 38% of lung cancers.
The claims have been taken seriously by
experts in the CRISPR field, but have not
caused undue alarm.
Associate Professor Marco Herold,
laboratory head at the Molecular Genetics of
Cancer Division of the Walter and Eliza Hall
Institute, Melbourne, is convinced CRISPR
remains a useful tool for applications in
research and the clinic.
“It has been known that DNA double