Dr Rafael J. Yáñez (or Yáñez-Muñoz)

Rafael J. Yáñez-Muñoz BSc PhD (Rafael Yáñez)

School of Biological Sciences, Royal Holloway-University of London, Egham, Surrey TW20 0EX, UK, e-mail: rafael.yanez@rhul.ac.uk

Rafael Yáñez is currently a Senior Lecturer in the Centre for Biomedical Sciences at the School of Biological Sciences, Royal Holloway-University of London, UK. Dr Yáñez previously held Lecturer appointments with King’s College London and University College London, and received his PhD and BSc in Biochemistry and Molecular Biology from the Autonomous University of Madrid, Spain. Dr Yáñez has a long-standing interest in gene therapy by both gene complementation and gene repair (homologous recombination). He is an expert in cell transgenesis and viral technology and has specifically researched on DNA viruses, therapeutic gene repair and viral vectors. Rafael Yáñez led the team that published in Nature Medicine the first in vivo demonstration of high transduction efficiency by integration-deficient lentiviral vectors (IDLVs), a very significant improvement in the bio-safety of this vector system. He is currently developing several viral vectors for further applications of research and clinical relevance, with particular interest in neurodegenerative and inherited diseases. Dr Yáñez is a member of the Board of the British Society for Gene Therapy and of two European Union FP7-funded consortia aiming at the development of gene therapy from bench to bed-side. He organises the largest UK event to mark Rare Disease Day (www.rhul.ac.uk/rarediseaseday)

Overview of current research

Our laboratory works on gene therapy for neurodegenerative diseases using novel, integration-deficient lentiviral vectors and adeno-associated viral vectors. We are particularly interested in the treatment of spinal muscular atrophy, Parkinson disease and stroke, and in exploiting endogenous neurogenesis. We are also using these vectors for gene repair in monogenic diseases including severe combined immunodeficiency and Duchenne muscular dystrophy. Another major goal is converting the non-replicating lentivector episomes into replicating episomes of wider applicability.

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Labelling of neurons with integration-deficient lentiviral vectors. A vector expressing eGFP (a gene that makes cells fluoresce green) was used to mark cells in the spinal cord (left) or the olfactory bulb in the brain (right). On the left panel motor neurons were also stained red with an antibody, so if these cells have taken up the viral vector the overlap of red and green fluorescence in their cell bodies is seen as yellow.

Why use non-integrating lentivectors?

Many gene therapy strategies require transduction (genetic modification with a viral vector) of somatic stem cells, neurons or other cells which divide rarely or do not divide at all. HIV belongs to a class (Genus) of viruses called Lentivirus, which in turn are part of a wider Family called Retroviridae, or more commonly, retroviruses. Lentiviruses distinguish themselves from other retroviruses in several ways, including their ability to cross the nuclear membrane, which allows them to infect cells that are not dividing. However, in common with other retroviruses, lentiviruses integrate their genome into the chromosomes of the cells they infect. Retroviral and lentiviral vectors likewise integrate into the genome of the transduced cells, which can lead to unwanted effects on the genes at or near the integration site, something called insertional mutagenesis. In the worst-case scenario such negative events can lead to cancer. Furthermore, each transduced cell will have the vector integrated at a different chromosomal location, which may affect or not vector gene expression. This can cause differences in vector gene expression in different cells, what we call position effect variegation. It has long been known that lentiviral vectors can be made integration-deficient using integrase mutations, but previously observed gene expression levels in vivo were very poor in the absence of integration.

Generation of episomal lentivector circles. The linear double stranded DNA vector molecule produced by standard lentivectors either integrates in the cellular genome or is converted into viral episomes. Higher levels of viral episomes are produced if integration is prevented through the use of mutations affecting the viral integrase.

Effective gene therapy with non-integrating lentivectors

We have recently demonstrated that lentiviral (HIV) vectors modified to prevent integration in the cellular genome are very efficient tools for gene therapy (Yáñez-Muñoz et al., 2006). We render the vectors integration-deficient by using missense mutations altering the integrase active site. Failing to integrate in the host cell genome these lentivectors generate increased levels of episomal vector circles, which lack replication signals and get diluted out through cell division. Gene expression from the viral episomes is transient in dividing cells but long-lived and efficient in quiescent tissues, including eye, brain, spinal cord and muscle (Yáñez-Muñoz et al., 2006; Fabes et al., 2006). The main advantages of these non-integrating lentivectors in gene addition strategies are their highly reduced risk of causing insertional mutagenesis and their avoidance of position effect variegation.

Effective gene expression and therapy with non-integrating lentivectors in vivo. (Left) Integration-defective lentivector encoding eGFP was injected subretinally in adult mice. The image shows eGFP fluorescence in the fundus of the eye 9 months post-injection. (Right) RPE65-encoding lentivector was injected subretinally in RPE65-deficient mice. The electroretinograph shows electrical activity in the retina of the treated eye (but not in the contralateral eye) three weeks post-injection, indicative of the prevention of retinal degeneration caused by RPE65 deficiency (Courtesy of Prof Robin Ali).

Other uses of non-integrating lentivectors

Lentiviral episomes can also be used as platforms for cassettes designed for site-specific or homologous recombination with the cellular genome. These strategies allow targeting of such cassettes to safe havens where no cellular genes will be negatively affected by the insertion event. Homologous recombination (gene targeting) can also be used for gene repair, the ideal form of gene therapy for inherited diseases (Yáñez and Porter, 1998, 1999, 2002a, 2002b). The development of designer nucleases, which can cut the target gene and thus greatly boost the frequency of homologous recombination, has been a determining event to make gene repair a credible therapeutic strategy. The inventors of gene targeting received the 2007 Nobel Prize in Physiology or Medicine.

Correction of a mutation by gene repair. A corrective vector carrying genomic DNA with wild-type sequence undergoes homologous recombination with the mutant gene, resulting in the correction of the genetic mutation (orange lollipop). The corrected gene is expressed under physiological regulation from its endogenous locus.

The episomal lentiviral circles do not have replication sequences and in proliferating cells they are progressively lost by dilution as the cell population expands. This makes them good vectors for transient gene expression in dividing cells, where they can provide a moderate expression level.

Transient gene expression by integration-deficient lentiviral vectors in proliferating cells. HeLa cells were transduced at the indicated multiplicity of infection (MOI, vector copies/cell) with integration-proficient (int+) or integration–deficient (int-) lentivector expressing eGFP. The percentage of green cells at the indicated times was determined by flow cytometry. Similar percentages of transduction were achieved at 3 days post-transduction regardless of integration proficiency. Transduction percentages with non-integrating vector decline progressively as the cell population expands.