GENETIC MODIFICATION OF HEMATOPOIETIC STEM CELLS: FROM BENCH TO BEDSIDE....AND BACK
Fulvio Mavilio
Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi, 287, 41100 Modena, Italy
Transplantation of genetically modified hematopoietic stem cells (HSCs)
is a potential therapy for a variety of genetic and acquired blood
disorders, such as severe combined immunodeficiencies (SCIDs),
thalassemia and AIDS. Recent improvements in stem cell culture and
vector technology are providing new tools for obtaining clinically
relevant numbers of genetically modified HSCs. A number of crucial
issues, however, remain unresolved, and need to be addressed for
genetic modification of HSCs to enter routine clinical practice.
Retroviral vectors provide the only available tool for inserting
therapeutic transgenes into human hematopoietic cells at efficiency
compatible with clinical applications. These vectors have been used in
hundreds of gene therapy trials since 1991, and were considered
relatively safe until the report of lymphoproliferative disorders
caused by insertional activation of the LMO2 proto-oncogene in two
X-SCID patients treated with retrovirally-transduced HSCs. MLV-derived
vectors were recently discovered to integrate preferentially around
gene promoters and transcription start sites, where the insertion of
LTR transcriptional enhancers is more likely to interfere with normal
gene regulation. However, no serious adverse event was reported so far
in clinical trials for other immunodeficiencies (ADA- SCID or CGD),
while evidence of oncogenic cooperation between LMO2 and the γc
lymphokine receptor, the therapeutic gene in the X-SCID trial, was
recently reported. This suggests the existence of disease-, protocol-,
and transgene-specific risk factors that may have contributed to the
relatively high frequency of malignancy in the X-SCID case. In-depth
analysis on the consequences of retroviral vector integration in other
clinical trials is therefore necessary, in order to provide
risk-benefit assessments in different biological and clinical contexts.
Research on new gene transfer system is also necessary, in order to
provide safer alternatives to retroviral vectors. HIV-1-derived
lentiviral vectors transduce human HSCs at high efficiency without
compromising self-renewal, repopulation and differentiation capacity.
Last-generation self-inactivating (SIN) vectors have minimized the
chances of generating replication-competent HIV derivatives during
packaging, and also potentially reduced the risk of insertional
oncogenesis. These vectors are now the most promising tools to
transduce human HSCs for gene therapy applications. Development of new
vectors aimed at targeted integration into the human genome is the goal
of much research in Europe and the U.S. We have recently described the
construction of hybrid vectors carrying the site-specific integration
machinery of the adeno-associated virus (AAV) in the framework of
high-capacity, helper-dependent adenoviral (Ad) vectors.
Drug-inducible, Rep-mediated integration of transgenes of intact size
was obtained in the AAV-specific site on chromosome 19 (AAVS1) in human
primary cells in culture, and in the liver of AAVS1 transgenic mice
upon a single, tail-vein administration of the vector. Non-random
integration of double-stranded DNA can therefore be obtained ex vivo
and in vivo by the use of hybrid Ad/AAV vectors with reasonable
efficiency, indicating a possible alternative to randomly integrating
RNA vectors for some gene therapy applications.
Key words:
gene therapy, retroviral vectors, insertional mutagenesis, genetic diseases
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