Tag Archives: gene therapy

From clinic to mouse to clinic: New HIV gene therapy shows promise!

Yesterday a team of University of Pennsylvania researchers – led by Dr Pablo Tebas, Professor Carl June, and Dr Bruce Levine – announced the successful conclusion of a clinical trial to evaluate the safety of a new gene therapy technique for treating HIV. It is a result that may eventually allow millions of HIV positive people to control the infection without having to take daily medication.

Two technicians in Penn Medicine's Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Two technicians in Penn Medicine’s Clinical Cell and Vaccine Production Facility hold up a bag of modified T cells. Image: Penn Medicine

Their study, published in the New England Journal of Medicine, involved taking a sample of T-cells from 12 patients and then using an adenoviral vector to introduce into these cells an enzyme known as a zinc-finger nuclease (ZFN) that has been targeted to the CCR5 receptor gene so that it introduces a mutation called CCR5-delta-32.  They then expanded the number of T-cells in vitro until they had billions of the transformed T-cells ready for transplant back into the patients.

Most HIV strains need to bind to CCR5 to infect T-cells, and the CCR5-delta-32 mutation prevents this binding and subsequent infection, as was dramatically demonstrated in the case of the “Berlin patient”, so the Pennsylvania team are hoping that their method will enable long-term control of HIV infection in patients, so that they may no longer need to take anti-retroviral medication.

An important part of the development of this therapy was its evaluation in vivo in an animal model of HIV infection. To do this they turned to mice rather than the more usual SIV/macaque model, as the sequence of the CCR5 gene at the site targeted by ZFN in macaques is not conserved with humans and would require the design and assembly of a distinct ZFN binding set for testing in SIV infection. Mice don’t normally become infected with HIV, but by using NOG mice that have been genetically modified so that their own immune system do not develop and then transplanting human immune cells into the mice they were able to produce mice with “humanized” immune systems that could be used to evaluate the ability of their ZFN modified T-cells to block HIV infection. In a paper published in the journal Nature Biotechnology in 2008, the team led by Carl June reported that the transformed human T-cells could successfully engraft and proliferate when transplanted into the NOG mice, and protect against subsequent HIV infection.

To our knowledge, genome editing that is sufficiently robust to support therapy in an animal model has not been shown previously. The ZFN-guided genomic editing was highly specific and well tolerated, as revealed by examination of the stability, growth and engraftment characteristics of the genome-modified sub-population even in the absence of selection…We also observed a threefold enrichment of the ZFN-modified primary human CD4+ T cells and protection from viremia in a NOG mouse model of active HIV-1 infection. As predicted for a genetically determined trait, the ZFN-modified cells demonstrated stable and heritable resistance in progeny cells to HIV-1 infection both in vitro and in vivo. These results demonstrate that ZFN-mediated genome editing can be used to reproduce a CCR5 null genotype in primary human cells.”

Following this they also undertook more extensive regulatory studies in mice to demonstrate that there were no toxicities associated with the ZFIN transformation of the T-cells.

While the clinical trial announced yesterday focused on the safety of the technique, the authors also reported that HIV RNA became undetectable in one of four patients who could be evaluated, and that the blood level of HIV DNA decreased in most patients, which bodes well for future trials when larger quantities of ZFN-modified cells will be transplanted.

This is not the first time that the pioneering work of Bruce Levine and Carl June has caught our attention, they are the same researchers who have hit the headlines with an innovative “Chimeric Antibody Receptor” gene therapy for leukemia that is part of the cancer immunotherapy revolution now underway. Their latest breakthrough is another indication of how gene therapy is becoming an important part of 21st century medicine.

Paul Browne

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Visionary Science: Gene therapy saves sight thanks to animal research

Yesterday the BBC News and Guardian Newspaper reported that a team led by surgeon Professor Robert Maclaren at the Oxford Eye Hospital had succeeded in using gene therapy to halt the decline in vision in six patients with the progressive eye disorder choroideremia.

All six patients were taking part in a clinical trial, and what was especially exciting was the sustained improvement in vision in the two patients whose vision had deteriorated the most. This is great news for the patients themselves, and as the technique is likely to be applicable to many different genetic eye disorders it is also good news for many millions of people who may benefit in future. It is also an excellent example of how years of research in mice, dogs and monkeys can lead to an important clinical advance.

Choroideremia is caused by a defect in the CHM gene, which encodes the Rab escort protein 1 (REP1), and lack of this protein leads to gradual degeneration of the retinal epithelium layer  (RPE) and rod photoreceptor cells in the eye, causing a progressive decline in vision that usually starts with night blindness and loss of peripheral vision, and eventually leads to total blindness.

To halt this decline Professor Maclean’s team used a vector  based on a modified adeno-associated virus serotype 2 (AAV2) which could express the healthy CHM gene in the eye and produce REP1.  Why did they choose AAV2 out of all the potential virus vectors available? The Lancet paper reporting on this trial cites a key study published in the Journal of Molecular Medicine in  2013* by Professor Maclaren and colleagues, which describes the development and evaluation of the vector used in the trial. In their introduction and discussion they discuss the rational for choosing the AAV2 vector:

With a functional fovea, safety with regard to avoiding a vector-related inflammatory reaction is of paramount importance. Two recent clinical trials had demonstrated that serotype 2 adeno-associated viral (AAV2) vectors have no long-term retinal toxicity when administered at the dose range 1010–1011 genome particles [12, 13]. Importantly, in addition to transducing the RPE, AAV2 is also known to target rod photoreceptors efficiently in the non-human primate [14], providing the ideal tropism for a CHM gene therapy strategy.

… Although one might argue that other serotypes such as AAV8 may be more efficient in targeting photoreceptors, AAV2 with the CBA promoter remains the gold standard for retinal transduction as evidenced by the sustained vision in Briard dogs treated with AAV2 vector over a decade ago [35].

12. Cideciyan AV, Aleman TS, Boye SL, Schwartz SB, Kaushal S, Roman AJ, Pang JJ, Sumaroka A, Windsor EA, Wilson JM, et al. Human gene therapy for RPE65 isomerase deficiency activates the retinoid cycle of vision but with slow rod kinetics. Proc Natl Acad Sci U S A. 2008;105:15112–15117. doi: 10.1073/pnas.0807027105.  13. Jacobson SG, Cideciyan AV, Ratnakaram R, Heon E, Schwartz SB, Roman AJ, Peden MC, Aleman TS, Boye SL, Sumaroka A, et al. Gene therapy for Leber congenital amaurosis caused by RPE65 mutations: safety and efficacy in 15 children and adults followed up to 3 years. Arch Ophthalmol. 2011;130:9–24. doi: 10.1001/archophthalmol.2011.298.  35. Bennicelli J, Wright JF, Komaromy A, Jacobs JB, Hauck B, Zelenaia O, Mingozzi F, Hui D, Chung D, Rex TS, et al. Reversal of blindness in animal models of Leber congenital amaurosis using optimized AAV2-mediated gene transfer. Mol Ther. 2008;16:458–465. doi: 10.1038/sj.mt.6300389.

So which two clinical trials are they referring to? Well, as you can see from the references they are referring to the successful trials of gene therapy for Leber Congenital Amaurosis (LCA)whose development we discussed on this blog back in 2009. As McLaren and colleagues point out, the sustained expression of RPE65 and long-term recovery of vision in the Briard dog model of LCA was a key factor in their decision.  The observation that AAV2 could be used to drive gene expression in rod photoreceptors was also important, as Maclaren and colleagues had previously generated a genetically modified mouse model of Choroideremia by knocking out CHM expression in the eye, and established that in Choroideremia the degeneration of rod photoreceptors is independent of the degeneration of the RPE, so it is crucial that the vector can drive healthy gene expressed in both the rods and RPE.

To develop the vector Maclaren and colleagues first compared the efficiency of 3 different promoters (promoters are sections of DNA that promote gene expression) -AAV2/2-EFS, AAV2/5-EFS and AAV2/2-CBA  – in driving expression of the CHM gene when added in vitro in a variety of dog and human fibroblast (connective tissue cell)  lines in an AAV2 vector, and then when injected in vivo in the retinas of healthy mice. These studies demonstrated that the most efficient AAV2 vector – named AAV2/2-CBA-REP1 – could drive expression of high levels of REP1 in both the RPE and rod photoreceptors of mice. After identifying the most effective AAV2 vector for expressing REP1  they assessed whether it was capable of expressing REP1 in isolated human retina’s obtained post-mortem from human donors, which it did. They then evaluated whether there as any toxicity associated with expressing REP1 in vivo in the retina of healthy mice, finding that AAV2/2-CBA-REP1 was non-toxic even when injected into the retina at high doses, and that it did not adversely affect vision.

Following these studies the question remained; would injection of AAV2/2-CBA-REP1 stop deterioration of vision in choroideremia?

To address this Maclaren and colleagues turned again to the genetically modified mouse model of choroideremia thay they had created earlier. Injection of the vector into the retinas of these CHM mice:

Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham injected eyes confirming that AAV2/2-CBA-REP1 is a promising  vector suitable for choroideremia gene therapy in human clinical trials.”

In other words the therapy worked in the mouse model of choroideremia, paving the way for the successful clinical trial reported this week.

This new therapy is another example of the importance of animal studies to the development of new clinical techniques and therapies, but also highlights the fact that medical science is a long game, with basic and applied research conducted more than a decade, even two decades,  ago being crucial to this week’s exciting announcement. This is something policy makers would do well to remember!

Paul Browne

* While this paper was published in 2013, the work it reports was completed several years earlier, before the clinical trial was launched in 2011.

1) Tanya Tolmachova, Oleg E. Tolmachov, Alun R. Barnard, Samantha R. de Silva, Daniel M. Lipinski, Nathan J. Walker, Robert E. MacLaren,corresponding author and Miguel C. Seabra “Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo”  J Mol Med (Berl). 2013 July; 91(7): 825–837. PMCID: PMC3695676

Successful gene therapy for hemophilia A in dogs – humans next!

On Wednesday we were saddened learned that double Nobel laureate Fred Sanger had died,  so it was fitting that yesterday also saw the announcement of an important scientific advance that owes everything to the molecular biology revolution he helped to launch – one that may improve the lives of many thousands of people with Hemophilia A.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia can affect dogs, and research on dogs with haemophilia has helped develop therapies for the disease. Image: Understanding Animal Research.

Hemophilia A is caused by a deficiency in the production of coagulation factor VIII, which leads to an increased risk of bleeding, and is due to defects in the gene located on the X-chromosome that lead to either insufficient production of factor VIII or production of defective factor VIII. Patients with severe Hemophilia A require frequent intravenous injections of recombinant factor VIII to prevent serious bleeding.  The BBC reported on Wednesday that a team of scientists based in the US and France have developed a gene therapy that successfully treated hemophilia in 2 dogs, and continued to prevent serious bleeds more than 2 years following treatment. Their therapeutic strategy involved isolating bone marrow hematopoietic stem cells (stem cells that give rise to all types of blood cell – including red blood cells, white blood cells and the platelets that are crucial to clotting) and transforming them with a lentiviral vector containing a gene encoding factor VIII under the control of a promoter that had previously been shown to drive expression of the drives the expression the target gene in the platelets of mice and dogs.  The transformed hematopoietic stem cells were then infused back into the same dog from which they had been isolated. Writing in the Nature Communications paper (1) reporting this study lead author Dr David Wilcox of the Medical College of Wisconsin and his colleagues discuss why dogs were the ideal subjects for preclinical evaluation of this therapy (for a great article on the crucial role of dogs in haemophilia research see this article from the magazine HEMAWARE).

 A canine model for haemophilia A exists, which results from a genetic mutation causing a large inversion of the FVIII gene (that resembles a molecular genetic defect found in about 40% of humans with the severe haemophilia A)4. Likewise, canine haemophilia A is essentially identical to the human disease in its clinical presentation characterized by severe-intermittent episodes of joint bleeding and haemorrhage. Protein replacement therapy is the most common treatment of severe bleeding episodes for haemophilia A but it has been confounded by the formation of inhibitory antibodies to transfused human FVIII in 30% of patients5,6. Similarly, 100% of dogs utilized from the Chapel Hill colony for this study develop inhibitory antibodies after being transfused with human FVIII (ref. 7), albeit severe bleeding is successfully treated with canine FVIII supplements. Thus, canine haemophilia A appears to be an ideal system to determine whether platelets can be used successfully to deliver human FVIII to the site of a vascular injury as a feasible approach to improve haemostasis within a ‘large-animal’ model of haemophilia A with the ability to form inhibitory antibodies to human FVIII.”

The above quotation also refers to an advantage of the technique they used over previous gene therapy methods developed to treat hemophilia A, one that the BBC article surprisingly didn’t pick up on. The BBC article mentions that an advantage that targeting expression of factor VIII to the platelets over previous studies where factor VIII was expressed in the liver of dogs with hemophilia A is that it would be suitable for patients who have damaged livers, but this is not the main advantage. The production of inhibitory antibodies against recombinant factor VIII by the patient is a problem that reduces the effectiveness of current therapies in about a third of people with hemophilia A, and this was also a problem in gene therapy techniques previously tested in clinical trials where factor VIII is secreted into the bloodstream from tissues such as the liver. Dr Wilcox and colleagues had the idea that by targeting expressing factor VIII specifically to platelets it would not be exposed to and blocked by inhibitory antibodies.

Laboratory Mice are the most common species used in research

GM mice are aiding the development of innovative therapies for many diseases, and haemophilia A is no exception.

The test this theory they turned to a genetically modified mouse model of hemophilia A, which had already proven very useful in earlier stages of the development of gene therapy to treating hemophilia A. In a study undertaken in GM mice (2) which had been immunized so that they produced inhibitory antibodies against human factor VIII, Dr Wilcox and colleagues at the Medical college of Wisconsin demonstrated that the their lentiviral vector that directed factor VIII expression specifically to the platelets resulted in the expression of therapeutic levels of factor VIII associated with platelets in the blood, even 6 months after treatment.

What’s more, they showed that this was possible using a nonmyeloablative conditioning regime before infusing the transformed hematopoietic stem cells. Conditioning regimes reduce the immune response to a transplant (and in cancers such as leukemia also eradicate the cancerous cells, using drugs such as the nitrogen mustards that we discussed earlier this week) but the myeloablative conditioning regimes that are very effective in treating leukemia carry significant risks, for example from infections following the procedure. Nonmyeloablative conditioning that does not completely destroy the patient’s reduces the risk of infection and transplant related death, and is thus more appropriate for conditions that are not immediately life threatening. This study paved the way for the evaluation of platelet specific factor VIII therapy in dogs that was reported on Wednesday. It is noteworthy that in the dogs, which were also treated using a nonmyeloablative pre-transplant conditioning regimen,  no inhibitory antibodies were detected against human factor VIII (unlike with previous gene therapy techniques), indicating that when associated with platelets it is sequestered from the immune system.

Almost 2 years ago we reported on the success of a small clinical trial of gene therapy in the treatment of hemophilia B following studies in mice and monkeys. We hope that with the development of a gene therapy technique that requires a milder conditioning regime and can avoid inhibitory antibodies this success will soon be repeated in hemophilia A.

Paul Browne

1)      Du LM, Nurden P, Nurden AT, Nichols TC, Bellinger DA, Jensen ES, Haberichter SL, Merricks E, Raymer RA, Fang J, Koukouritaki SB, Jacobi PM, Hawkins TB, Cornetta K, Shi Q, Wilcox DA. “Platelet-targeted gene therapy with human factor VIII establishes haemostasis in dogs with haemophilia A.” Nat Commun. 2013 Nov 19;4:2773. doi: 10.1038/ncomms3773. Pubmed 24253479

2)      Kuether EL, Schroeder JA, Fahs SA, Cooley BC, Chen Y, Montgomery RR, Wilcox DA, Shi Q. “Lentivirus-mediated platelet gene therapy of murine hemophilia A with pre-existing anti-factor VIII immunity.” J Thromb Haemost. 2012 Aug;10(8):1570-80. doi: 10.1111/j.1538-7836.2012.04791.x.PubMed 22632092. PMC3419807

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Italian Gene Therapy Success – Thanks to Animal Research

Italian scientists announced yesterday that they had successfully treated the deadly genetic disorders metachromatic leukodystrophy and Wiskott-Aldrich syndrome in a small clinical trial, marking and another major landmark in the developing field of gene therapy. As you might expect the breakthrough has received widespread media coverage, including CBS, the BBC and the Italian edition of the Huffington Post. While these reports do a reasonable job of explaining the significance of these two small trials, they do miss out one important fact, that this advance rests on many years of basic and applied scientific research, much of which required animal studies.

Many of the reports highlight the fact that this trial – led by  Dr Luigi Naldini, Dr Alessandra Biffi and Dr Alessandro Aiuti of the San Raffaele Telethon Institute for Gene Therapy - involved the use of parts of HIV virus to create a vector that can then insert function versions of the genes that are missing or defective into bone marrow stem cells of the patients, before transplanting the corrected cells back into the patients. This utilisation of a deadly virus to create a therapy that saves lives still takes people by surprise, but really it should not as this is not the first time. Last year we discussed on this blog how such a lentiviral vector had been used to successfully treat acute lymphoblastic leukemia while back in 2009 we saw how a similar gene therapy vector was used to treat the neurological disorder cerebral X-linked adrenoleukodystrophy (X-ALD). Animal research made crucial contributions to these two therapies, and it wasn’t surprising to find that it played an equally crucial role in developing gene therapy for metachromatic leukodystrophy and Wiskott-Aldrich syndrome.

In the paper published online in Science (1) reporting on the outcome of the trial of gene therapy in 3 boys with Wiskott-Aldrich Syndrome (WAS) , the Telethon team write that:

We developed a SIN lentiviral vector coding for human WASP* under the control of a 1.6 kb reconstituted WAS gene promoter (LV-w1.6W) (3). The use of this endogenous promoter ensures that the transgene is expressed in a physiological manner (4), restoring WASP expression and function in human and murine WAS cells (3, 30–34). Its moderate enhancer activity combined with the SIN LTR design reduces the risk of insertional mutagenesis (35), as shown by in vitro transformation assays (36) and preclinical in vivo studies in WASP-deficient mice (34, 37). These data provided the rationale for a phase I/II clinical trial in which LV-w1.6W was used as a gene therapy vector for treatment of patients with WAS.

*WASP, a protein that regulates the cytoskeleton and mutated in the syndrome

An open-access paper published earlier this year in the journal Molecular Therapy (2) describes the role of studies undertaken in immunodeficient mice to develop and evaluate the lentiviral vector to treat Wiskott-Aldrich Syndrome in more detail.

In the Science paper (3) reporting the results of the trial of gene therapy in 3 boys with metachromatic leukodystrophy, the Telethon team highlight the key contribution of studies in mouse models of metachromatic leukodystrophy (MLD) in evaluating the ability of hematopoietic stem cells (HSCs) transformd using the lentiviral vector containing the functional arylsulfatase A  gene – deficient in MLD – to treat the disease:

In a mouse model of MLD, we have demonstrated that disease manifestations can be prevented and corrected by lentiviral vector (LV)–based HSC-GT but not by HSCT (8, 9, 16). This is consistent with the observation that HSCT fails to provide consistent benefits in MLD patients (3, 5–7). LV–based HSC-GT induced extensive and supra-physiological expression of the functional ARSA gene throughout the HSC progeny, which in turn mediated widespread cross-correction of CNS and PNS resident cells (8, 9).

This was only the last in a long series of animal studies that led to the clinical trial, an open-access review of progress being made in the development of gene therapy for a range of leukodystrophies published by Dr Biffi in 2011 highlights not only this important preclinical work, but also the basic and translational studies undertaken using a variety of different vector types in mice and monkeys that provided the data that allowed scientists to develop an effective therapy.

At a time when science and medicine in Italy is under attack from charlatans who are promoting dubious stem cell therapies, and scientific activists are campaigning against laws that threaten the very future of medical research in Italy, this weeks good news from the San Raffaele Telethon Institute for Gene Therapy is a reminder that there are many excellent scientists in Italy who are conducting medical research at the highest level, and that their work depends on animal research. We hope that this breakthrough – and there can be no doubt that this is a breakthrough – heralds a better future for science in Italy.

Speaking of Research

1) Aiuti A. et al. “Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome” Science. Published online 11 July 2013, DOI: 10.1126/science.1233151

2) Scaramuzza S. et al. “Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.” Mol Ther. 2013 Jan;21(1):175-84. doi: 10.1038/mt.2012.23.

3) Biffi A. et al. “Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy” Science. Published online 11 July 2013, DOI: 10.1126/science.1233158

4) Biffi A, Aubourg P, Cartier N. “Gene therapy for leukodystrophies.” Hum Mol Genet. 2011 Apr 15;20(R1):R42-53. doi: 10.1093/hmg/ddr142.

Defeating Leukemia: A smile that says “Thank the mice”

A couple of days ago the New York times published a heart warming story about a young girl named Emma Whitehead whose acute lymphoblastic leukemia – which had previously defied all therapies – has gone into full remission following treatment with a novel gene therapy that programmed her immune system to target the cancer cells. The New York Times report noted that the therapy used a vector based on the HIV-1 virus to deliver genes – known as a chimeric antigen complex (CAR) – to modify  Emma’s T-cells so that they would destroy the leukemia cells.  This isn’t the first example of how scientists are using the properties of this deadly virus to develop powerful new therapies, back in 2009 we discussed how such a lentiviral vector was used to treat the genetic disease cerebral X-linked adrenoleukodystrophy. Emma wasn’t the only patient to benefit from this therapy developed by scientists at the University of Pennsylvania, 9 other patients with intractable leukemia have experienced partial or full remissions.

Emma2

Earlier today I received an e-mail from a long-time reader of this blog asking:

Did I dream there was an SR post on this already?”

Well my friend, you were not dreaming.

Last year we published a post entitled “A breakthrough against Chronic Lymphocytic Leukemia…thank the mice!” which discussed the role of animal research in the development of this therapy, and in particular that of mice the evaluation of chimeric antigen complexes in order to identify a complex that would induce a long-lasting immune response against the cancer cells. Our post also linked to an article on the Weizmann Wave Blog entitled “Cancer Breakthrough 20 Years in the Making” which described the basic biomedical research – mice were again crucial – that underpinned this field.

At the time I concluded the post by saying:

So there you have it, behind the headlines are years of graft by hard-working and innovative scientists, who utilised a wide range of experimental approaches – among which animal studies figure prominently – to develop a novel therapy for CLL.”

And I say the same again today. At a time when funding of medical research in the US is facing the threat of very damaging cuts, Emma’s story is a reminder of why you should write to your Senator and Congressional Representative today!

Paul Browne

New gene therapy for mitochondrial diseases a step closer thanks to ONPRC

Mitochondria are fascinating. These tiny organelles that reside within almost all of the cells in our bodies (mature red blood cells being an exception) generate the supply of a molecule called adenosine triphosphate (ATP) which is the principle source of energy that cells, and ultimately ourselves, need to survive. They also have an intriguing evolutionary history, being descended from bacteria that over one and a half billion years ago formed a symbiotic relationship with primitive eukaryotic cells that are the ancestors of today’s plants and animals. A legacy of this ancestry is that animal mitochondria contain a tiny genome that encodes 37 genes that are crucial to the mitochondria’s function, and separate from the main genome which is found in the nucleus of the cell and contains just over 20,000 coding genes. Unlike nuclear genes, half of which are inherited from our mother and half from our father, mitochondrial genes are almost always inherited from the mother only, which means that if a mother has a mitochondrial genome mutation it will always be passed on to her children.  However, since a human egg cell contains many mitochondria, and only some of them may be defective, there is usually a threshold level of defective mitochondria which must be reached before the defects cause disease in children, and the severity of disease can be very variable. Nevertheless inherited mitochondrial disorders affect as many as 4,000 children born in the USA every year, and for almost all of them treatment options are limited.

One way in which the transmission of mitochondrial diseases can be prevented is by screening embryos during IVF, and earlier this year we reported on how a team at the Oregon National Primate Research Centre (ONPRC) led by Dr. Shoukhrat Mitalipov discovered through studies performed on Rhesus macaques how to improve the efficiency of this screening. However in cases where screening does not identify eggs that are free from mitochondrial genetic defects other ways of preventing transmission of the disorders are being examined, and one of these is the possibility of replacing the damaged mitochondria with healthy mitochondria from a donor.

Yesterday in a publication in Nature (1), Dr. Mitalipov’s team at ONPRC announced another major advance made possible through research on Rhesus monkeys, the first demonstration that it is possible to replace the faulty mitochondria of a human egg cell before fertilization and create healthy looking human embryos, from which embryonic stem cells could be derived that were identical to controls created through normal IVF.

Mitochondrial Gene Therapy. Source Mitalipov Lab/OSHU

Briefly, the procedure involved the removal of the nuclear genetic material from the egg of a patient whose mitochondrial DNA contains mutations, and its transplantation into an egg containing normal mitochondrial DNA from which the nuclear genetic material has been removed.  More detailed descriptions and discussion of the process used in this therapy and the team’s results can be found on the Oregon Health and Science University website, reports on the BBC and LA Times, and in Nature News, it’s clearly been a study that has caught the imagination of a lot of people!  It’s worth noting that a child born after fertilization with the partner’s sperm would be free of risk from maternal mtDNA mutations as well as being the biological child of the patients, since the mitochondrial genome accounts for only 37 of over 20,000 coding genes in the body it is inaccurate to refer to these as 3 parent embryos.

The news reports make it very clear that research on monkeys was crucial to this advance, indeed the potential of the technique used – which they term spindle–chromosomal complex transfer – was first demonstrated when they were able to produce 3 healthy monkey infants in 2009 (2).  They started by examining the distribution of Rhesus monkey mitochondria during the process of meiosis – the type of cell division through which gametes (sperm and egg cells) are produced – using confocal laser scanning microscopy, and observed that at a particular late stage in the process termed the  metaphase II stage the mitochondria were distributed relatively uniformly throughout the cytoplasm,  except immediately around the chromosomes and the spindle apparatus (a protein structure segregates chromosomes between daughter cells during cell division), which were devoid of mitochondria.  This suggested that it might be possible to isolate the spindle–chromosomal complex at this stage and transfer it to an egg cell from which the egg had been removed without transferring any mitochondria at the same time.

A significant challenge was how to avoid damaging the spindle-nuclear complex during this operation, but the team had recently developed new techniques to transfer the nuclei of adult skin cells from monkeys into egg cells and successfully derive embryonic stem cells from the resulting clones.  By modifying these techniques they were able to reconstructed eggs that were capable of being fertilized normally, undergoing embryo development and producing healthy offspring. Genetic analysis confirmed that nuclear DNA in the three infant macaques originated from the spindle donors whereas mitochondrial DNA came from the cytoplast donors. This set the stage for the work announced yesterday.

In addition to reporting the production of human embryos through spindle–chromosomal complex transfer (ST) this week’s Nature paper (1) also reported the outcome of follow-up examination from birth to 3 years of four monkeys born through ST – the 3 reported in 2009 and one born subsequent to that publication – and found that they were developing normally and were in good health.

Because egg cells only remain viable for a short period of time after they are harvested from a donor, it is considered crucial that ST can be performed successfully using frozen egg cells for this technique to be clinically viable, so the team also examined if it was possible to do this using thawed Rhesus macaque cells. They were successful; the experiment resulted in the birth of a healthy monkey. More surprisingly they also found to their surprise that while the spindle–chromosomal complex could withstand prior cryopreservation the technique failed when the egg into which the spindle-chromosal complex is transferred had been frozen – indicating that most of the damage to the frozen egg is to its cytoplasm, rather than to the nucleus as had previously been thought, a discovery that may have wider implications for the future improvement of human egg cryopreservation and IVF techniques.

Impressive as this study is it is by no means the end of the road, this technique needs further refinement and optimization before anyone should attempt to use it in the clinic, but it does provide both scientists and ethicists with very valuable information.  This is particularly true in the UK, where the Human Fertilisation and Embryology Authority is reviewing this technique and another that is being developed by Professor Mary Herbert at the University of Newcastle. Speaking to the BBC yesterday, Peter Braude, Professor of Obstetrics and Gynaecology at King’s College London, said:

It is exactly the sort of science that the HFEA expert committee recommended needed doing, and demonstrates further the feasibility of this technique.”

We at Speaking of Research congratulate Dr. Mitalipov and his team at ONPRC on their groundbreaking work.

Paul Browne

1)      Masahito Tachibana, Paula Amato, Michelle Sparman, JoyWoodward, Dario Melguizo Sanchis, Hong Ma, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Eunju Kang, Hyo-Sang Lee, Cathy Ramsey, Keith Masterson, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard Stouffer& Shoukhrat Mitalipov “Towards germline gene therapy of inherited mitochondrial diseases” Nature Published online 24 Oct 2012, doi:10.1038/nature11647

2)      Masahito Tachibana, Michelle Sparman, Hathaitip Sritanaudomchai, Hong Ma, Lisa Clepper, Joy Woodward, Ying Li, Cathy Ramsey, Olena Kolotushkina & Shoukhrat Mitalipov “Mitochondrial gene replacement in primate offspring and embryonic stem cells” Nature 461, 367-372 (2009) doi:10.1038/nature08368