Retroviral vectors in gene therapy – is it the end of the road?

by Alexey Bersenev on April 3, 2008 · 12 comments

in Journal club, leukemia

About 10-15 years ago retroviral vector was one of the promising gene delivery systems in preclinical models of gene therapy because of its ability to integrate a gene of interest into the genome and maintain stable gene expression. Hematopoietic Stem (or progenitor) Cells (HSPCs) have been considered to be an ideal target for gene-correction of hematological and immune diseases, linked to particular gene or blood lineage defect.

Most clinical applications have used gene transfer vectors based on integrating retroviruses, but the relative inefficiency of these vectors has limited the considerable potential of gene transfer into HSPCs. Genetically modified cells represent only a small fraction (1%–10%) of the hematopoietic cells after transplantation. Consequently, competition from unmodified infused and endogenous HSPCs may dilute any therapeutic effect of the transduced cells. This implies that this small fraction of genetically modified hematopoietic cells will correct diseases requiring more than 1%–10% of corrected cells only if they have a marked selective growth advantage in vivo.

Short-term outcome of the clinical trials started in France in 2000 had shown some benefits for young patients with X-linked severe combined immunodeficiency – (SCID).

But what happened later?

The strong enhancers contained in retroviral genomes can activate adjacent cellular genes following integration, but the risk posed by one or a few integrated vectors per cell was estimated to be very low. However, these assumptions were shattered in 2002, when French researchers announced that a child who had shown unequivocal clinical benefit in the HSPC gene therapy trial for X-linked SCID (SCID-X) developed a vector-related T cell acute leukemia three years after infusion of retrovirally modified CD34+ cells. Since then, three other children in the French trial and, more recently, one patient in a similar SCID-X study conducted by British researchers have developed leukemias.

At the same time, researchers were looking for an efficient and safe HSPC expansion agent. Transcription factor HOXB4, which has been investigated in the last few years, has become one of the most promising HSPC expansion agents. HOXB4 significantly increases hematopoietic stem cell self-renewal and expansion and promotes hematopoietic differentiation from embryonic stem cells. It was shown that a retrovirus which contains HOXB4 dramatically increased HSPC engraftment without leukemia development in mice and human. Also, a recent study demonstrated that overexpression of HOXB4 in embroynic stem cell-derived HSPCs leads to enhanced engraftment and recovery of functional immunity without any tumorogenesis or leukemogenesis. Therefore, HOXB4 was considered as a promising gene target to expand HSPCs.

However, a recent study published in the Journal of Clinical Investigation has added “fuel to the fire” and broken all of promises made by potentials of retroviral therapeutics. A group of authors from Fred Hutchinson Cancer Research Center (Seattle, WA, USA), for the first time, investigated a long-term outcome of HSPC transplantation, by overexpressing HOXB4 with retroviral vector in large animals. 2 out of 2 dogs and 1 out of 2 macaques developed myeloid leukemia approximately 2 years after transplantation! In contrast, researchers did not see any sign of leukemia in the animals that were tranduced with a control vector that carried another gene (MGMT).

Here is an interesting question: was overexpression of HOXB4 itself the only single event that caused leukemia? Not really.

The new findings indicate the cooperativity between HOXB4, a growth-altering transgene, and transgene insertion sites. This cooperation event was quite similar to what researchers reported in failed SCID-X trials. Even though the study highlights that the gene HOXB4 is a catalyst for leukemogenesis, it also reminds us of a potential problem with viral gene transfer.

This picture demonstrates mechanisms of leukemia development after transplantation of HSPCs transduced with retroviral vectors:


In my opinion, gene therapy is in crisis. Researchers should come up with a new virus-free system for an effective gene delivery. Adeno-associated virus (AAV)-based therapy didn’t show any adverse effects so far, but who knows what we will get in upcoming preclinical and clinical studies?

At the same time we always have to think about the balance between a level of HSPC expansion ex vivo and a risk of cancer. Almost all regulators of normal hematopoiesis, which have been proposed to be good targets for stem cell expansion in clinical settings, were shown to be evolved into leukemogenesis when their expression levels were modulated. The implication is that one should not judge quickly whenever a large expansion rate of blood-forming stem cell is observed in laboratory. Besides, do we really need that many cells? It would be of uttermost importance to consider the patient safety first before thinking about any therapeutic potential of ex vivo HSPC expansion.

I would like to finish by an excellent quote from Cynthia E. Dunbar who has commented on this study:

The message of the current study by Zhang and colleagues is clear as a bell: overexpression of HOXB4 using integrating retroviral vectors is much too risky to be contemplated in any clinical settings. This is not to say that this study rules out alternative approaches to harnessing the power of HOXB4 on HSPCs.

Events in the recent history of gene therapy have led many to acknowledge that the field may have been, on several occasions, too eager to rush into the clinic. However, the wisdom gained from these setbacks is now becoming apparent in studies such as that of Zhang and colleagues in this issue of the JCI. Scientists and clinicians hoping to use their protocols to treat humans understand the importance of examining vectors’ behavior in a variety of comparative assays, including large animal studies, before entering the clinic. The use of well-characterized and well-tested preclinical models such as those of Zhang and colleagues is likely to minimize the risk of an adverse event that would throw the field into another crisis.

J. Clin. Invest. doi:10.1172/JCI34371

Commentary: Andre Larochelle and Cynthia E. Dunbar HOXB4 and retroviral vectors: adding fuel to the fire.

Very interesting blog post opinion (highly recommend to read!):
is gene therapy going down (or do we just play dumb?)

Read more:
Gene Therapy safety issues arise again and again

Geneticist Dr D Kohn with a five month old Apache baby who suffers from SCID (severe combined immunodeficiency). The baby is receiving gene therapy for its condition. It is isolated in a sterile tent to prevent infection. The rare genetic mutation of SCID destroys the immune system making the body unable to fight infection. SCID babies lack a vital enzyme which their immune system needs. Gene therapy involves inserting a gene for this enzyme into stem bone marrow cells and transplanting the cells into the baby. With this enzyme, stem cells may produce normal immune system blood cells. Photographed at the Childrens Hospital in Los Angeles, USA.(SciencePhotoLibrary)
PS: special thanks to Jae-Won Shin

{ 12 comments… read them below or add one }

steppen wolf April 4, 2008 at 7:55 am

Thanks for linking. That post of mine is just an opinion, obviously, but sometimes I do wonder whether some researchers set themselves up for failure (in terms of therapeutic value) just to publish a few more papers.

Anyway..nice blog you have here!


steppen wolf April 4, 2008 at 8:01 am

Alexey, I forgot to tell you…you will be hosting the next Cancer Research Blog Carnival, as per your request. I think the guys over at BayBlab still have to add you as a host (you might want to remind them by e-mail), but you should be able to make an account with in the meantime – you will need it anyways…


Alex April 4, 2008 at 8:18 am

to Steppen –
it’s a great opinion! love it completely :)))
more popular and in indie- style compared with mine, it’s so easy to read. I agree with your opinion.

Actually, if you look at back to history of clinical trials, it’s a usual thing when after start of trials (which have a rational – excellent preclinical data and FDA approval), adverse effects come up later.
That’s what we see now in gene therapy and in cell (stem cell) therapy as well.

But this is a thing about trials: we don’t know how it’s will going in human! This is experiment, even after FDA approval and safety tests.

Other side of coin – desperation of patients, who will sign of participation list agreement in any trial, because they have no choice (only experimantal new treatment) and rush of researchers and clinicians in the same time.

I’d agree with Cynthia Dunbar, that frequently we are too eager to jump to clinic. We have to be more careful about new methods. Translation to clinic is always hard.


sergey April 6, 2008 at 11:37 pm

Didn’t get the message.
1. Retrovirus caused leukemia after GT to blood cells – it’s known. 2. Retrovirus caused leukemia because of a huge viral dosage- it’s also known from publication and since Paracelsius times: Poison is just a question of dose 3. HOXB4 nicely expands HSPCs including those with retrovirally induced oncogene expression – again was known.
Are they crazy those guys that mixed poison and enhancer and was waiting for Lord to make them wise?


Alex April 7, 2008 at 9:46 pm

to sergey –
HOXB4 – very nice HSC expansion agent, yes, but it was unknown that it can cause leukemia. Even together with retrovirus, leukemia came up, the role of HOXB4 is prior and catalytic.

so conclusion 1: HOXB4 is not that safe what was considered before

Quote 1:

It has been demonstrated that high-level HOXB4 expression, achieved with retroviral (5) or adenoviral (15) vectors in human CD34+ cells, perturbed the myeloid differentiation program both in vitro (5, 15) an in vivo (5) without frank leukemia.

so = controversy 1

conclusion 2:

This study underscores the importance of large animal models in further development of gene and other novel HSPC-directed therapies.

Even we got to know about adverse effect of retrovirus-based gene therapy from human trials (unfortunately), this work has a great impact to the gene/cell therapy field.

It was long-term work and done during last 3 years.
I’d definitely allow to publish this in the journal of JCI level.


sergey April 7, 2008 at 11:55 pm

No problems, I just didn’t get a mess. 2 bad things together were supposed to neutralise each other? What was initial idea? Have you read 5 and 15? me not. Did those guys before they start? doubt. Large animal model? Bullshit, even with large you can be fine but fail on humans and vice versa. It’s not a point, not an idea, not even a prove. Regulations are working that ‘sit nothing scientific to write about. Bullshit.


Jae-Won Shin April 9, 2008 at 7:39 am

Good article and comments.. the major question is what now? so what? To me, one solution of this issue is to do things in parallel. This is especially important in therapeutics. Rather than relying on just retroviral vectors, one should also consider using any available small molecules, antibodies or siRNA in parallel as a therapeutic carrier to target HOXB4.

Another solution is to use an inducible form of transgene. I don’t know this so correct me if I am wrong. However, I don’t seem to understand why people do not consider an inducible form of promoter when introducing a gene. For example, doxicycline inducible promoter can be used to introduce it along with a transgene of interest so that only patients who are taking doxicycline can express this transgene. If this turns out to be dangerous, patients stop taking it so that transgene is not expressed anymore. Also, transgene can be ‘floxed’ so that cre can be used in an emergency situation to remove transgene from the body.

If a therapeutic method is potentially dangerous, one should either ditch it, or find a way to neutralize the danger. And do something about it. Simple is that.


Alex April 9, 2008 at 9:44 pm

to sergey –
doesn’t regulation organization’s decision to start phase I in human based on preclinical data?
I’d agree with thesis about mouse or large animal models – doesn’t really matter, because we never know what we will get in human after start the trial.
Why they are (virus + expansion agent) 2 bad things? Integration of gene of interest is good and expansion of HSC is good – that’s what we want! Problem is overlapping and became too much of everything. So we got a bad thing as a result.

Few protocols in parallel – good approach:
non-viral gene delivery?
other HSC expander?
small molecules?
going to read this

I don’t know about others but I learned a few things from this work,
for example I wouldn’t try to use HOXB4 as an therapeutic agent in human


Alex April 10, 2008 at 9:13 pm

I just don’t understand how those trials got approval in France and UK? If specialists know that retrovirus specifically integrate into genome near the oncogenes, what lead to their activation and subsequently to cancer. It is very well known, Sergey is right, so why?
Now FDA is kind of scared of pushing forward embryonic stem cell therapy, could be the same story.

Several years ago, patient deaths in gene therapy trials caused the FDA to halt all such trials under its jurisdiction, another consultant told me, and that field has never recovered.


sergey April 10, 2008 at 9:31 pm

Alex, It ‘s well known about retovirus adverse effects. That it may cause expression of protooncogenes in hematopoietic cells including stem. It was also known that HOXb4 could expand hematopoietic cells including stem and as an evident consequence including the one with protooncogene(s)induced. It s logically, well these guys got money for big animals, not bad, but nothing really exciting. As for the control somewhat called MGMT which frankly I do not know and which perhaps doesn’t affect nothing including cell expansion in a small group did nothing. Great!!! It s real success of these guys, use nonsense get nothing!! Reliable but stupid science. But… money back guarantee
Currently only viral delivery of the TRANSGENE is efficient, doesn’t matter floxed or hoxed.


Alex April 10, 2008 at 9:54 pm

to sergey –
ok, only viral delivery, so why trials with retroviruses were permitted? why not adeno- or AAV?
If gene therapy so risky why don’t we switch to hematopoietic cell transplantation for treatment of mono-gene defect diseases, at least associate with abnormal hematopoiesis.


Alex May 31, 2008 at 3:02 pm

Results of a new study (=gene therapy safety assay) from U California Davis published in Molecular Therapy showed that retroviral HSC gene/cell therapy is absolutely safe in mice model.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months . Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied . In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2–6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

For me it’s shows again how results of preclinical testing could be different between species and animal models. Again, even so, we never know what we will get in human when we will start a clinical trial.


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