Here we demonstrate, using both biological and physical assays, that overexpression of SOD2 substantially protects both murine bone marrow and K562 cells from ionising radiation injury..
Trang 1Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
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HEMATOLOGIC-NEW ADVANCES
>= 16.5 months after transduction Thus, direct intramarrow
administration of rSV40s yields efficient gene transfer to rat BM
progenitor cells, and may be worthy of further investigation
Retroviral Expression of Manganese Superoxide
Dismutase
Thomas D Southgate,1 Victoria Sheard,1 Michael D Milsom,1
Rob J Mairs,2 Marie Boyd,2 Leslie J Fairbairn.1
1 Gene Therapy, Paterson Institute for Cancer Research,
Manchester, United Kingdom; 2 Radiation Oncology, Cancer
Research UK Beatson Laboratories, Glasgow, United Kingdom.
Overexpression of manganese superoxide dismutase (MnSOD,
also known as SOD2) has been hypothesized to provide haemopoietic
cells with radioprotection through the scavenging of oxygen radicals
induced by ionizing radiation Here we demonstrate, using both
biological and physical assays, that overexpression of SOD2
substantially protects both murine bone marrow and K562 cells
from ionising radiation injury For this study the human SOD2
cDNA was cloned within a retroviral vector (SFβ91), optimised for
initiation of transcription in primitive haemopoietic cells, along with
eGFP as a marker Retroviral producer lines were established and
used to infect murine bone marrow and the human erythroleukaemic
cell line, K562 Cells were characterised by Western and Southern
blotting and shown to be polyclonal by LAM PCR SOD enzymatic
activity was assessed using a water-soluble tetrazolium salt
microplate assay, whilst survival was determined by assessing colony
forming ability after irradiation of cells at various doses The levels
of DNA strand breaks were assessed using a COMET assay
Cells transduced with the SFβ91-SOD2-eGFP vector exhibited
expression of SOD2 by Western blot analysis, eGFP by FACS and
a 2.5 fold increase in total SOD enzymatic activity In SOD2
transduced murine bone marrow and in K562 cells we have
demonstrated a physical decrease in the levels of DNA strand breaks,
whilst biologically we showed an approximately 2-fold survival
advantage in SOD2-expressing cells over controls when exposed to
varying doses of ionising radiation In conclusion, overexpression of
SOD2 using a retroviral vector to infect cells of the haemopoietic
compartment delivers both physical and biological protection against
ionising radiation Our findings are encouraging for the development
of SOD2 gene transfer for the protection against myelotoxic side
effects of radiation
Transporter Variant Does Not Effect Proliferation
or Differentiation of Hematopoetic Stem Cells
Olga Ujhelly,1 Nora Kucsma,1 Judith Cervenak,1 Linping Chen,2
Manuel Grez,2 Balazs Sarkadi,1 Katalin Nemet.1
1 Experimental Gene Therapy, National Medical Center, Institute of
Haematolgy and Immunology, Budapest, Hungary; 2
George-Speyer-Haus, Institute for Biomedical Research, Frankfurt,
Germany.
Stem cell based gene therapy is often unsuccessful because of the
low number of gene modified cells Growth advantage of the corrected
cells may provide permanent recovery of the patient The multidrug
resistance phenotype of haematopoietic stem cells (HSCs) allows
the selection of gene-modified cells in vivo or in cancer patients,
decreases the myelosuppressive side effect of chemotherapy In
contrast with the potential benefit, some authors reported a harmfull
side effect of several ABC transporters on the myeloid differentiation
In the present work, overexpression of a substrat mutant of human
ABCG2 (ABCG2-482G) was investigated in a mouse model
Animals were transplanted with bone marrow cells transduced by a
retroviral vector encoding ABCG2-G Colony forming properties
(CFU-C) of the transduced cells were investigated in vitro, while
the repopulation of haematopoiesis was followed for 16 weeks Bisides ABCG2 expression, chimerism and linaege markers were detemined
Our results show a highly detectable (45-60%), functionally active population of ABCG2-G positive progenitor cells, still the number
of CFU-C was similar in the treated and control sample No significant difference was found in the hemopoietic tissues (bone marrow, spleen) nor in the mature white blood cell linaeges (WBC)
of the transplanted mice
Our data suggest that ABCG2 contribute in the protection of essential tissues and does not influance hematopoesis
ABCG2-482G is an ideal candidate for gene therapy application as an in vivo
selectable marker
Cells with Non-Viral Gene Transfer
Meenakshi Noll,1 Ryan Bennett,1 Stephen Yant,2 Mark A Kay,2
1 Dept of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR; 2 Dept of Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA.
The life span of patients with Fanconi Anemia (FA) is severely shortened by bone marrow failure and malignancies and currently, allogeneic bone-marrow transplantation is the only therapeutic option Despite the successful modeling of FA viral vector gene therapy in the mouse, application of the same approach to humans
is complicated by the requirement for prolonged ex vivo manipulation and the scarcity of HSCs in this disease Here, we utilized the non-viral Sleeping Beauty (SB) transposon system to facilitate stable, gene transfer of the human FANCC cDNA without ex vivo culture This was achieved either by electroporation of naked DNA into whole bone marrow or by direct injection into the femur cavity Our studies demonstrate that a) hematopoietic stem cells from Fancc -/
- mice can be transduced by plasmid DNA; b) stable integration of plasmid DNA into the genome of hematopoietic stem cells (HSC) is possible; c) animal model was phenotypically corrected; and d) long-term therapeutic benefit could be achieved using this method Non-viral gene transfer may be a viable alternative for the treatment
of hematopoietic diseases
Retroviral Expression of the X-Ray Cross Complementing Protein (XRCC1)
Thomas D Southgate,1 Katherine Clarke,1 Leslie J Fairbairn.1
1 Gene Therapy, Paterson Institute for Cancer Research, Manchester, United Kingdom.
Administration of the chemotherapeutic camptothecin, a topoisomerase I inhibitor, causes a dose-limiting toxicitiy to rapidly renewing tissues leading to myelosuppression, diarrhoea and hemorrhagic cystitis Although the use of camptothecin deriviates, such as topotecan has reduced the urological toxicity associated with this class of compounds, they all still exhibit a marked toxicity
to myeloid progenitors
We have investigated whether retroviral transfer and expression
of the x-ray cross complementing protein (XRCC1), using a vector (SFß91) which is optimised for transcription in primitive haemopoetic cells, could protect the haematopoietic compartment from camptothecin damage Although lacking any direct DNA repair activity, XRCC1 acts as a scaffolding protein facilitating the repair
of both direct DNA strand breaks (such as those induced by camptothecin) and indirect DNA strand breaks (caused by agents such as the halogenated thymidine analogues)
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GENE REGULATION: REGULATED SYSTEMS AND TISSUE SPECIFIC EXPRESS
Here we demonstrate that cells transduced with SFß91 XRCC1
IRES eGFP acquire a 6 fold increased resistance to camptothecin
and 5-iodo-2’-deoxyuridine and display increased kinetics of repair
Resistance to these agents could be reversed by addition of either
the PARP inhibitor 3-aminobenzamide, or methoxyamine (an
inhibitor of base excision repair) respectively Furthermore, cells
transduced with XRCC1 demonstrate a significant increase in
protection against ionising radiation In summary,
retrovirally-mediated transfer and expression XRCC1 to cells of the primary
haemopoietic compartment delivers both physical and biological
protection against the action of agents that cause either direct or
indirect DNA strand breaks Our findings are encouraging for the
development XRCC1 gene transfer for protection against the
myelotoxic side effects of chemotherapy using camptothecin or its
derivatives and we are currently evaluating this therapy in in vivo
toxicity models
Homing Endonucleases for Gene Correction
Applications
Andrew M Scharenberg,1 David J Rawlings,1 Raymond J
Monnat,3 Barry L Stoddard.2
1 Pediatrics, University of Washington, Seattle, WA; 2 Basic Science,
Fred Hutchinson Cancer Research Center, Seattle, WA; 3 Genome
Sciences, University of Washington, Seattle, WA.
Induction of a double strand break in DNA results in a large
increase in the frequency of homologous recombination in the vicinity
of the double strand break, raising the possibility of targeted gene
correction if reagents were available for the simultaneous introduction
of a site specific double strand break and a correcting DNA fragment
Structural and molecular studies of homing endonucleases (HE’s)
suggest that HE’s possess significant potential for engineering of
their cut site specificities to diverse target sequences Here we
discuss preliminary work on the I-AniI endonuclease with regards
to its application to targeted gene correction of primary immune
deficiencies Work to be discussed will focus on the engineering
hurdles to be faced in applying I-AniI as a site specific DSB-inducing
tool, and the development of model systems for the evaluation and
benchmarking of HE’s and other DSB-inducing reagents in practical
gene correction methods
Blood Hematopoietic Cells Expansion
Kamran Alimoghaddam,1 Mitra Khalili,1,2 Masoud Soleimani,3 Lili
Moezi,1 Ardeshir Ghavamzadeh.1
1 Hematology, Oncology and BMT Research Center, Tehran
University of Medical Sciences, Tehran, Islamic Republic of Iran;
2 Molecuar Biology, Khatam University, Tehran, Islamic Republic
of Iran; 3 Hematology, Tarbiat Modaress university, Tehran,
Islamic Republic of Iran.
Objectives: to find the best cell culture condition for expansion of
cord bloods hematopoietic CD34+/CD38- ex vivo expansion and
study the role of MIP/a in expansion potential
Material and methods: CD34+/CD38- cells separated from cord
bloods by Mini-MACS and cultured in different culture mediums
including 50ng/ml of TPO,IL-6,SCF and flt3-ligand.for some samples
we used RPMI+ 10% FCS or autologous cord blood plasma and for
others we used serum free media(SF) Also we added MIP/a
(10-50ng/ml) to some samples We cultured the cells for 2 weeks in CO2
incubator and studied expansion potential by counting of MNCs
,CFU-assay , LTC-IC and studied the number of
CD34+/CD38-cells before expansion 7 days and 14 days after expansion
Results: expansion potential of cord blood hematopoietic cells was good and maximally expanded to 25 times Also we found that serum free media isbetter than FCS 10% and autologous cord blood plasma MIP/a did not changed expansion potential of hematopoietic cells
Conclusion: serum free media is the best medium for expansion and from GMP points of view and MIP/a is useful for expansion to prevent maturation during expansion that may be useful for increasing transduction efficiency without induction of maturation
Expression in the Medial Forebrain Bundle: A Reversible Model of Dopamine Depletion
Mary E Garrity-Moses,1 Qingshan Teng,1 Jun Yang,1 Thais Federici,1 Erin Gilbert,2 Thyagarajan Subramanian,2 Nicholas M Boulis.1
1 Neurosurgery, The Cleveland Clinic Foundation, Cleveland, OH;
2 Neuroscience, The Cleveland Clinic Foundation, Cleveland, OH.
We have previously demonstrated focal synaptic inhibition through neuronal expression of the light chain (LC) fragment of tetanus toxin
in vivo The transient effects are spatially discrete lending them to
application in deep brain nuclei This experiment examines feasibility
in creating a rat model for Parkinsons disease through gene-based synaptic inhibition of the substantia nigra The present experiment examined the impact of nigral LC expression on apomorphine induced rotations
Methods: Tetanus light chain (LC) was cloned into an adenoviral
vector under control of the CMV promoter containing a GFP marker Next, the impact of unilateral nigral 6-OHDA on striatal dopamine and glutamate synapses was compared to unilateral nigral LC expression Rats received medial forebrain bundle (MFB) injections
Apomorphine-induced rotational behavior was assessed using a rotometer weekly for up to 4 weeks
Results: A significant increase in contralateral rotation was
observed in the 6-OHDA positive control group and the 8µL TeTxLC group, in comparison to the 4µL TeTxLC and PBS groups 6-OHDA animals demonstrated an average of 7.84 rotations per minute (+/-0.45 SEM) and rats receiving 8µL TeTxLC demonstrated an average
of 4.39 rotations per minute (+/- 0.41 SEM) PBS rats demonstrated
an average of 0.325 rotations per minute and rats receiving 4µL TeTxLC demonstrated an average of 0.708 rotations per minute
Significance: This initial model proves the feasibility of
dopamine depletion through nigral LC expression Because LC expression inhibits synaptic activity without killing neurons, this approach represents a strategy for transient dopamine depletion A subsequent experiment will apply an adeno-associated vector containing a Tet-on expression cassette (rAAV.Tet-on.LC) This latter vector will facilitate controlled, transient nigral suppression and will facilitate the study of behavioral recovery and normalization of striatal receptors following the recovery of striatal dopaminergic input Transient and controlled nigral inhibition may provide a superior model for studying striatal recovery and dopaminergic re-innervation