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Improving the efficiency of gene targeting in blood stem
cells


Image courtesy of Wikimedia

As mentioned in the
introductory post
to this series on blood stem cell gene
therapy, several significant issues existed with the first set of
clinical trials in the gene therapy world. It became abundantly
clear that simply delivering the gene target on its own would not
be sufficient for curing disease. Rather, these early trials
inspired a great need to investigate the human body’s response to
the gene delivery by viruses, how to improve the efficiency of gene
delivery, and how to avoid the nastiness of off-target effects.

The human body has developed many ways to fight off viruses and
it should come as no surprise that hijacking viruses as a delivery
vehicle for nucleic acids runs the risk of activating some of these
mechanisms. For readers interested in a more comprehensive
description, I would suggest an
academic review
from Shirley et al.; and, for those interested
in a lay summary Wikipedia
summarizes
the hurdles reasonably well.

The Shirley review goes through several commonly used gene
therapy viral vector strategies and will be a much more accurate
read than my summary that follows. Briefly, adenovirus vectors used
in early trials were selected for their incredibly high
transduction efficiency and subsequent expression of the target
gene. Unfortunately, this high expression was capable of triggering
a massive immune response and soon represented one of the greatest
risks for applying gene therapy in clinical practice. This led to
the exploration of alternative approaches with lower efficiencies,
but less immunological stimulation.

One of the most effective strategies was modifying the vectors
to contain fewer, and eventually no, viral gene components. This
led to the development of adeno-associated
vectors
, or AAVs, which have become a mainstay of gene
therapy.

These vectors do not carry any viral gene coding sequences and
consequently do not drive a strong immune response compared to
their parental adenovirus vectors. A large number of current
approved gene therapies use AAVs, although they do not perform well
in targeting cells in vitro and are generally not considered to
integrate in the genome (potentially risking not having the
long-term stable expression required for successful gene therapy).
(N.B., native AAV can integrate
in the genome)

Minimizing the off-target problem

To achieve long-term stable expression, many gene therapy
developers have opted to utilize lentiviral vectors (derived from a
parental HIV virus and able to deliver a gene to non-dividing blood
stem cells). The flip-side of stable expression via genome
integration is the very issue that caused massive problems of gene
therapy induced cancer in the X-SCID trial in the early 2000s
described in my introductory post. Unpredictable and continued
integration of the genetic material delivered in the therapy can
lead to disruption of cancer initiating genes (called
“insertional mutagenesis”) with obvious undesired
consequences.

Second and third generation vectors have greatly minimized this
risk by removing elements of the virus that are essential for viral
re-integration. This effectively means that after the virus
initially integrates, it stays put and does not move around the
genome again. The formal possibility of being unlucky in that first
integration still exists, but removing the ability of the virus to
replicate in vivo has dramatically
improved
the safety profile of lentiviral approaches and has
been widely adopted.

Treating the not-so-straightforward genetic
disorders

While numerous gene therapies are working well for relatively
straightforward single-gene disorders (i.e., Wiskott-Aldrich
Syndrome), there are a number of diseases that require quite a lot
more development due to the complicated nature of the gene(s)
involved. Two of these blood disorders, sickle cell disease and
beta-thalassemia, predominantly affect lower-middle income
countries and the genetics are complicated. A recent
paper
from one of the world’s foremost experts in gene
therapy, John Tisdale, highlights some of these complications:

“Unlike those used in other diseases, therapeutic vectors for
the β-globin diseases have demanding requirements including
high-level β-globin expression, tissue specificity among erythroid
cells, long-term persistence, and high-level modification at the
HSC [blood stem cell] level.”

These blood stem cells are the foundations of durable gene
therapy: without introduction of the corrected gene into blood stem
cells, the transplanted cells will have a finite lifespan and are
highly unlikely to lead to a curative treatment.

As discussed in the
second post of this series
, increasing
blood stem cell numbers would be one method of addressing this
concern, but in the case of β-globin disorders, it is critical to
have a high proportion of corrected hemoglobin molecules, so
targeting efficiencies are of utmost importance to improve. A
number of trials are underway in
sickle cell disease in particular
.

In summary, while gene therapy does seem to have made some
incredible strides in the past few years, there is an enormous
amount of work remaining to address the more complicated, and in
this case substantially more wide-reaching, genetic diseases. Much
of this work still exists at the technical level. Trying to get the
right gene correction, delivered in the right number of cells, in
the correct proportion, will continue to challenge researchers in
this exciting area of science.


Image courtesy of Wikimedia

As mentioned in the
introductory post
to this series on blood stem cell gene
therapy, several significant issues existed with the first set of
clinical trials in the gene therapy world. It became abundantly
clear that simply delivering the gene target on its own would not
be sufficient for curing disease. Rather, these early trials
inspired a great need to investigate the human body’s response to
the gene delivery by viruses, how to improve the efficiency of gene
delivery, and how to avoid the nastiness of off-target effects.

The human body has developed many ways to fight off viruses and
it should come as no surprise that hijacking viruses as a delivery
vehicle for nucleic acids runs the risk of activating some of these
mechanisms. For readers interested in a more comprehensive
description, I would suggest an
academic review
from Shirley et al.; and, for those interested
in a lay summary Wikipedia
summarizes
the hurdles reasonably well.

The Shirley review goes through several commonly used gene
therapy viral vector strategies and will be a much more accurate
read than my summary that follows. Briefly, adenovirus vectors used
in early trials were selected for their incredibly high
transduction efficiency and subsequent expression of the target
gene. Unfortunately, this high expression was capable of triggering
a massive immune response and soon represented one of the greatest
risks for applying gene therapy in clinical practice. This led to
the exploration of alternative approaches with lower efficiencies,
but less immunological stimulation.

One of the most effective strategies was modifying the vectors
to contain fewer, and eventually no, viral gene components. This
led to the development of adeno-associated
vectors
, or AAVs, which have become a mainstay of gene
therapy.

These vectors do not carry any viral gene coding sequences and
consequently do not drive a strong immune response compared to
their parental adenovirus vectors. A large number of current
approved gene therapies use AAVs, although they do not perform well
in targeting cells in vitro and are generally not considered to
integrate in the genome (potentially risking not having the
long-term stable expression required for successful gene therapy).
(N.B., native AAV can integrate
in the genome)

Minimizing the off-target problem

To achieve long-term stable expression, many gene therapy
developers have opted to utilize lentiviral vectors (derived from a
parental HIV virus and able to deliver a gene to non-dividing blood
stem cells). The flip-side of stable expression via genome
integration is the very issue that caused massive problems of gene
therapy induced cancer in the X-SCID trial in the early 2000s
described in my introductory post. Unpredictable and continued
integration of the genetic material delivered in the therapy can
lead to disruption of cancer initiating genes (called
“insertional mutagenesis”) with obvious undesired
consequences.

Second and third generation vectors have greatly minimized this
risk by removing elements of the virus that are essential for viral
re-integration. This effectively means that after the virus
initially integrates, it stays put and does not move around the
genome again. The formal possibility of being unlucky in that first
integration still exists, but removing the ability of the virus to
replicate in vivo has dramatically
improved
the safety profile of lentiviral approaches and has
been widely adopted.

Treating the not-so-straightforward genetic
disorders

While numerous gene therapies are working well for relatively
straightforward single-gene disorders (i.e., Wiskott-Aldrich
Syndrome), there are a number of diseases that require quite a lot
more development due to the complicated nature of the gene(s)
involved. Two of these blood disorders, sickle cell disease and
beta-thalassemia, predominantly affect lower-middle income
countries and the genetics are complicated. A recent
paper
from one of the world’s foremost experts in gene
therapy, John Tisdale, highlights some of these complications:

“Unlike those used in other diseases, therapeutic vectors for
the β-globin diseases have demanding requirements including
high-level β-globin expression, tissue specificity among erythroid
cells, long-term persistence, and high-level modification at the
HSC [blood stem cell] level.”

These blood stem cells are the foundations of durable gene
therapy: without introduction of the corrected gene into blood stem
cells, the transplanted cells will have a finite lifespan and are
highly unlikely to lead to a curative treatment.

As discussed in the
second post of this series
, increasing
blood stem cell numbers would be one method of addressing this
concern, but in the case of β-globin disorders, it is critical to
have a high proportion of corrected hemoglobin molecules, so
targeting efficiencies are of utmost importance to improve. A
number of trials are underway in
sickle cell disease in particular
.

In summary, while gene therapy does seem to have made some
incredible strides in the past few years, there is an enormous
amount of work remaining to address the more complicated, and in
this case substantially more wide-reaching, genetic diseases. Much
of this work still exists at the technical level. Trying to get the
right gene correction, delivered in the right number of cells, in
the correct proportion, will continue to challenge researchers in
this exciting area of science.

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