Biochemical Society Transactions ( 1999) Volume 27, part 5
c4
Herpes virus vectors for gene therapy in the nervous system
l2shdmm
Windeyer Institute of Mediul Sciences, University College London,
Windeyer Building, Cleveland Street, London W I P 6 0 8
A I 37
C6 Wfdte heart allogmft s w i v d after &novirus-mediated
gene lmnsfer of E F P l or CXK.44.
Lgnacio Amgon. INSERM W37.30, Bv. J. Monnei 44093, N a n e h.
email:
[email protected].
Many of the most intractable human diseases such as Alzheimer’s and
Parkinson’s diseases involve the nervous system making a gene therapy
approach to such diseases very attractive. Although a number of viruses
have been proposed as vectors for gene delivery to the nervous system,
herpes simplex virus (HSV) has unique advantages. Thus, it is easy to grow
and manipulate in culture, naturally establishes asymptomatic infections of
the nervous system and has a large genome size allowing multiple genes to
be inserted. The use of this virus as a gene delivery vector however,
requires two key problems to be overcome. These are firstly the effective
disablement of the virus so that it does not cause damage in the brain but
still retains its gene delivery ability and secondly the need to achieve long
term expression of the inserted gene. I will discuss the progress made in
overcoming these two difficulties so as to produce a vector which may
ultimately be of use in human gene therapy procedures. I n addition, I will
discuss the use of these viruses carrying specific genes to either reduce
rotational behaviour in a rodent model of Parkinson’s disease in the central
nervous system or to reduce neuronal cell death following sciatic nerve
damage in the peripheral nervous system.
Administration of certain Th2 cytokines, TGFPl or CTLA4, has been
implicated in the establishment of tolerance in transplantation models.
We constructed recombinant adenoviruses (Ad) containing either rat
IL-4, a cysteine 223- and 225-mutated form of simian TGFPl
(AdTGFP, second generation vector) or mouse CTLA4Ig (AdCTLA4,
first Peneration vector) and confirmed their expression with bioassays
and mRNA analysis. These molecules were expressed in rat cardiac
grafts after direct myocardial injection of the adenoviruses at
transplantation, using the MHC-incompatible combination of Lewis
I W (RTIw) donors and LEW.IA (RTI a) recipients. Successful cardiac
gene transfer was confirmed by RT-PCR, using primers specific for
adenoviral IL-4, TGFPl or CTLA4Ig and injection of an Ad coding
for lacZ (AdlacZ). Similar graft survivai times were observed in
untreated hearts (6.8M.5d, n=10) and hearts injected with varying
doses of either AdlacZ (10.7+2.3d, n=4), Ad lacking the expression
cassette (Add1324) (9.2*1.2d, n=8) or AdIL-4 (7.8fl.3d. n=6).
Hearts injected with AdTGFP had significantly prolonged allograft
survival (>97+15.7d, n=13, p<O.OOOl), with 5/13 grafts showing > I 30
days survival. Recipients transplanted with AdTGFP-transduced hearts
and treated with anti-TGFP antibody showed graft survival times
similar to the controls (14.8M0.8d, n=5). Gene transfer with
AdCTLA4 resulted in indefinite survival in all cases (n=14).
Immunohistological analysis of the AdTGFP- and AdCTLA4-treated
grafts showed decreased numbers of T cells and IL-2R+ cells,
respectively. Compared to the controls, the grafts transduced with
AdTGFP showed significantly reduced levels of IFNy and T N F a
mRNA, whereas AdCTLA4-transduced grafts showed reduced levels
of IFNy and increased levels of IL-13. Longstanding LEW.IA
recipients (hearts transduced with either AdTGFP or AdCTLA4)
rejected LEW.1W skin allografts with the same kinetics as third-party
donors. AdTGFP long survivor grafts showed minimal signs of
chronic rejection with no evidence of major fibrosis, whereas
AdCTLA4 long survivor grafts showed moderate signs of rejection.
These results demonstrate that Ad-mediated gene transfer of TGFPl
or CTLA4 can significantly prolong the survival of adult cardiac
allografts through various mechanisms, resulting in different
outcomes.
C5
C7
Towards Gene Therapy for Cystic Fibrosis
Christopher F Higgins, Stephen C. Hyde and Deborah R. Gill
MRC Clinical Sciences Centre, Imperial College School of Medicine,
Hammersmith Hospital, Du Cane Rd., London W12 ONN, U.K.
Tel: +44-181-383-8335: Fax: +44-181-383-8337; email:
[email protected] and
Nuffield Department of Clinical Biochemistry,
University of Oxford
John Radcliffe Hospital
Oxford OX2 9DU
0 1999 Biochemical Society
IMMUNOTHERAPY OF AUTOIMMUNE DISEASES
BY GENE TRANSFER
w,
David Gould. Alex Annenkov, Hanna Drejal. Gordon Daly.
Gabriel A. Rabinovich2. Ludovic Croxford3. David Baker3, Manana Berenstein
4and Osvaldo Podhajcer4
Bone and Joint Research Unit. St. Bartholornew’s and Royal London School of
Medicine and Dentistry, Queen Mary and Westfield College, Charterhouse
Square, London W C l M 6BQ, Kennedy Institute of Rheumatology. Londonl,
Departamento de Inmunologia, Universidad de Cdrdoba2; Institute of
Ophthalmology, London3; Fundacidn Campomar, Buenos Aires4
Autoimmune diseases are characterized by both a cellular and a humoral
pathogenic component. T cells secrete cytokines and help B cells to
differentiate and mature into antibody secreting cells. Certain antibody
isotypes such as IgG2a in the mouse fix complement and augment the
damage to tissues by direct cytotoxicity or by recruitment of inflammatory
cells due to the production of the anaphylatoxins C5a and C3a. Some
cytokines are proinflammatory (such as TNF and IFNy) are produced by Th 1
T cells. Other anti-inflammatory cytokines produced by Th2 cells (such as
IL-4. IL-10)or Th3 cells (TGFP) can down regulate both antigen presenting
cells and Thl cell function. The effects of Thl cytokines, can be also
suppressed by natural antagonists such as soluble cytokine receptors.
Delivery of anti-inflammatorycytokines such as TGFB, IFNP or cytokine
inhibitors such as soluble TNF-R. We have used retroviral vectors with
inhibitory cytokines or cytokine-inhibitors to transduce either nonmigrating syngeneic fibroblasts, or mobile pathogenic T cells. We have
shown therapeutic effect in animal models of arthritis and multiple
sclerosis. We have provided evidence that local injection of cells or
complexes of cationic liposomes with mammalian expression plasmids
into the central nervous system can control experimental allergic
encephalomyelitis. Some immuno-modulators such as galectin-1, which
induce apoptosis of T cells cannot be expressed constitutively in
lymphocytes as i t induces their death. However, expression of galectin-l in
fibroblasts implanted intraperitoneally was extremely efficient at down
regulating arthritis. The immune mechanism s involved in the therapeutic
effect showed that all the genes mentioned above affect both T cell function.
as measured by cytokine profile expression, as well as affecting the humoral
compartment assessed by a down regulation of anti-collagen antibody
production mainly a reduction in IgG2a production.