Clinical gene transfer had its official beginning when, in 1989, 5 patients with terminal melanoma were given autologous lymphocytes that had been "marked" ex vivo with a gene encoding resistance to the antibiotic G418. This study was designed primarily to trace the cells in the patients' bodies and to show the safety of gene transfer, and in that sense it was successful. No helper viruses were found, no reverse transcriptase activity was detected, no toxicity was experienced, and the transduced cells remained otherwise "normal"ref.
:
(INGN 201 / Ad-p53 / Ad5CMV-p53 / Adenoviral p53 / INGN 101 / RPR/INGN
201ref1,
ref2
(Advexin®; source : Introgen's ) has 18 patents) : the first
randomised phase II/III trial testing this approach in patients with ovarian
cancer was closed early because the treatment did not provide adequate
therapeutic benefit : dominant negative "cross talk" between p53 mutants
that are overexpressed in ovarian cancer, coupled with aberrant silencing
of genes, may have compromised the effectiveness of p53 gene therapyref.
On the other side a advanced non-small lung cancer was treated by a local
injection of ADVEXIN once every 4 weeks for 14 times without marked adverse
events, which resulted in tumor regression and relief of his symptom for
a yearref,
and B7-1 genes (Ad-p53/GM-CSF/B7-1)
cells
cells via enhanced apoptosis and the immunogenicity of tumor cells was
augmented. The combinatorial effect of these 3 genes on inducing cytotoxic
T lymphocytes (CTLs) was more evident than that of p53 individually or
any combinations of 2 (p53 + GM-CSF or p53 + B7-1). Furthermore, significant
proliferation of autologous peripheral blood lymphocytes (PBLs) and specific
cytotoxicity against autologous primary MM cells were induced in vitroref
(low levels of gene expression are required, whereas in diseases with dominant
heredity very high levels of gene expression are required, ie > 50% of
the wild-type homozygote expression level !). E.g. Tyr
hydroxylase in Parkinson's
disease (PD)
: CTLA4Ig fusion gene delivery and expression in vivo using retrovirally
transduced (using the centrifugal enhancement method) myeloid dendritic
cells that induce alloantigen-specific T cell anergy in vitro by
blocking expression of CD80 and CD86, costimulatory signals necessary for
naive
T cell activationref
after BMT...).
: NO.
is an hypoxic cell radiosensitizer. Spontaneous
and bioreductive NO. donors
can't administered in sufficient doses to achieve tumor radiosensitization
in the clinic as they elicit systemic vasodilation and hypotension.
(toxigenic approach)
were generated by placing the region encoding the mature plant ribosome-inactivating
protein under the control of CMV or SV40 promoters. Their ability to inhibit
protein synthesis was first tested in cultured tumor cells co-transfected
with a luciferase reporter gene. In particular, SAP expression driven by
CMV promoter (pCI-SAP) demonstrated that only 10 ng of plasmid per 1.6
x 104 B16 cells drastically reduced luciferase activity to 18%
of that in control cells. Direct intratumoral injection of pCI-SAP complexed
with either lipofectamine or DOTAP in B16 melanoma-bearing mice resulted
in a noteworthy attenuation of tumor growth. This antitumor effect was
increased in mice that received repeated intratumoral injections. A SAP
catalytic inactive mutant (SAP-KQ) failed to exert any antitumor effect
demonstrating that this was specifically owing to the SAP N-glycosidase
activity. The gene encoding SAP, owing to its rapid and effective action
and its independence from the proliferative state of target cells might
become a suitable candidate suicide gene for oncologic applicationsref
to increase radioactive iodide (131I)
uptake (RAIU). In fact it is generally believed that the therapeutic
effectiveness of 131I for patients with thyroid cancer largely
depends on the unique ability of thyroid tissues to organify 131I
(increaseable by co-delivery of thyroid
peroxidase (TPO)). Prolonged 131I retention is thought to
be essential to achieve sufficient therapeutic dose. However, therapeutic
dose delivered to tumors could also be increased by increased 131I
uptake and the use of alternative radionuclides with more potent radioactivity
(e.g. 188ReO4).
It has been proved for medullary
thyroid carcinomaref|
|
|
|
| CB1954 (5-aziridin-1-yl-2,4-dinitrobenzamide), SN24927 | Escherichia coli aerobic or Bacillus amyloliquefaciens nitroreductase (NTR), mammalian Walker DT-diaphorase (DTD) [+ NAD(P)H] | 2-hydroxylamine; 4-hydroxylamine ==non-enzymatic reaction with cellular thio-esters==> bifunctional alkylating agent |
| 5-fluorocytosine (5-FC / 5-FCyt / flucytosine) | bacterial cytosine deaminase (bCD, usually from Escherichia coli)) or yeast CD (yCD, usually from Saccharomyces cerevisiae). CD is absent in mammalian cells. | toxic anabolite 5-fluorouracil (5-FU / 5-FUra) == cellular enzymes of rapidly proliferating (normal or tumor) cells ==> 5-fluoro-UTP (FUTP) and 5-fluoro-dUMP (FdUMP) : the former is incorporated into RNA and interferes with RNA processing, while the latter irreversibly inhibits thymidylate synthase and hence DNA synthesis. Tumour-selective amplification of the intracelular anabolic activation of 5-FU to FUMP can be achieved by transfection with the Escherichia coli gene for uracil phosphoribosyltransferase (UPRT) |
| 6-methyl-9-(2-deoxy-b-
D-erythro-pentofuranosyl) purine (6MEPDR) |
Escherichia coli purine nucleoside phosphorylase (PNP) | 6-methylpurine (6MEP) |
| fludarabine
phosphate |
2-fluoroadenine (2FA) : the compound can be incorporated into both RNA and DNA, killing non-dividing, as well as dividing cells. 2FA is 100-fold more potent as a growth inhibitor than 6MEP. | |
| 6-thioxanthine (6TX) | Escherichia coli XGPRT (gpt) | |
| ganciclovir (GCV)
(Cerepro®; source : Ark
Therapeutics) |
HHV-1
/ HSV-1-tk
(the equine herpes virus 4 tk is a better suicide gene than the HHV-1 tkref)
: GCV phosphate transfer to non-HSV-TK-expressing bystander cells may mediate
either bystander cell killing or sparing of HSV-TK-positive cells, depending
upon the cell specific drug metabolismref. |
GCV-monophosphate => cellular enzymes => GCV-triphosphate (kills dividing cells by acting as chain terminator) |
| acyclovir (ACV) |
acyclovir monophosphate | |
| tricyclic purine nucleoside analogues (TPNA) : lower affinity
for HSV-tk; cytostatic potency and selectivity as for bicylclic
compounds; more pronounced bystander effect in cell culture at relatively
high drug concentrations; intrinsically strong fluorescent properties which
allow simple and sensitive monitoring; higher lipophilicity => better uptake
from the blood into CNS.
1) tricyclic ACV derivative   |
||
| 1-[3-(2-bromoethylamino)-2-
hydroxypropyl]-2-nitroimidazole (RB6145) ==in vivo hydrolysis ==> 1-[3-aziridinyl-2-hydroxypropyl]- 2-nitroimidazole (RSU1069) |
flavoprotein NADPH cytochrome P450 (c) reductase under the control
of a hypoxia-responsive
element (HRE)
(O2-sensitive GDEPT) |
highly reactive toxic species which can be back-oxidized to the non-toxic parent compound only in the presence of O2 |
| cyclophosphamide (CP) |
CYP2B6 |
4-OH-cyclophosphamide, that breaks imidazole ring in G leading to depurination of GMP residue. |
| indole-3-acetic acid (IAA) | horseradish peroxidase (HRP) | |
| ethanol |
alcohol dehydrogenase (ADH) |
acetaldehyde (AcH)ref |
),
ensuring that it is not necessary to transfect all of the cells in the
tumour. Fusion of TK to an 11-amino acid peptide from the basic domain
of HIV-1
Tat protein (Tat11)
imparts cell membrane-translocating ability to the enzyme and significantly
increases its cytotoxic activityref1,
ref2.
The efficacy of this strategy was tested in 2 different mouse models of
adoptive tumorigenesis, based on the implantation of human Kaposi sarcoma
(KS-IMM) cells in nude mice or of B16F10 melanoma cells in syngeneic C57BL/6J
mice. Experiments were performed by the subcutaneous injection of mixtures
of unmodified tumor cells containing different fractions of TK or Tat11-TK
producing cells followed by animal treatment with ganciclovir. In both
systems mice bearing tumors containing Tat11-TK cells displayed significantly
retarded tumor growth and prolonged survival as compared with mice inoculated
with cells expressing unmodified TK. Collectively, these results demonstrate
that fusion of Tat11 to TK imparts remarkable intercellular trafficking
capability to the enzyme. This modification of TK might constitute an important
step in the optimization of TK suicide gene strategy for gene therapy of
cellular proliferationref
imparts cell membrane translocating ability to the enzyme and significantly
increases its cytotoxic efficacy. In cells expressing CPP-TK, this protein
is found extracellularly, associated with cell surface heparan sulfate
proteoglycans (HSPG), and is released into the cell culture medium. Based
on its interaction with HSPGs, the protein is then internalized by neighboring,
nonexpressing cells, amplifying bystander effect by another route.
before transplantation
might provide a means of permitting the ablation of these cells if they
subsequently misbehave (malignancy in any tissue that retains growth potential,
immune
pathology
on the part of lymphocytes (e.g. donor T cells for GvHD, ...), or hyperfunction
on the part of cells intended to supply a missing function at regulated
levels). Particular concerns may apply to ESCs
because of evidence of their epigenetic instability. Ionizing
radiation (IR)-responsive promoters
can be used to combine radiotherapy
and gene therapy.
genetically engineered with AAV
vector are attracted by tumors as they use these cells for tumour
neoangiogenesis
(just like they migrate to injuries to repair wounds) and continue to develop
and grow in the tumor environment, representing an effective delivery system
for gene therapy in pediatric soft-tissue sarcomas
M-55 (Mose strain Cl. oncolyticum M55)ref
strain VNP20009 is a genetically modified strain possessing an excellent
safety profile, including genetically stable attenuated virulence (a deletion
in the purI gene), reduction of septic shock potential (a deletion
in the msbB gene), and antibiotic susceptibility. The combination
of the lipid mutation and the purine auxotrophy attenuate the virulence
of the bacteria by greater than 10,000-fold and enhance the specificity
of the bacteria for tumor tissue (accumulate in tumor tissue at levels
at least 200-fold and, more commonly, 1000-fold greater than in other normal
tissues : colonization of normal tissues gradually decreases with timeref).
In mice, VNP20009 is rapidly cleared from the blood from a peak level of
1x104 cfu/mL to undetectable levels in 24 h. In nonhuman primates,
VNP20009 was also rapidly cleared from the blood, from a peak level of
1.0x106 CFUs/mL to undetectable levels in 24 h. VNP20009 was
detected in the liver, spleen, and bone marrow of monkeys; the amount decreased
over time, and VNP20009 was cleared from all organs by day 41; no VNP20009
could be detected in the urine or feces of the monkeys. VNP20009 is genetically
stable after many generations of growth (> 140) both in vitro and in
vivoref.
These bacteria have been found to be safe in mice, pigs and monkeys when
administered intravenously. 4-5 days after a single IV injection of 1 x
106 CFUs/mouse, we routinely detected VNP20009 proliferation
and accumulation at levels ranging from 1 x 108 to 2 x 109
CFUs/g tumor. The amount of VNP20009 accumulated in the liver ranged from
3 x 104 to 2 x 106 CFUs/g. The distribution of Salmonella
in tumors was homogenous; YS7212 could be detected from the periphery to
the interior portion of the tumors. Bacteria accumulation and CD expression
persisted in the tumors for up to 14 days after a single bolus IV administration
of bacteria to tumor-bearing miceref.
Optimal therapeutic cose (OTD) was found to be 5 x 107 CFUs/rat.
In humans by the intratumoral route, a maximum tolerated dose has not been
reached, and dose escalation continues past the current dose level of 4
x 107/m2. Furthermore, VNP20009 persisted in injected
tumors for at least 2 weeks in 8/11 patients treated to date. By 30-min
IV administration, a maximum tolerated dose (MTD) of 3 X 108
CFUs/m2 has been established. The maximum-tolerated dose was
3 x 108 CFUs/m2. Dose-limiting toxicity was observed
in patients receiving 1 x 109 CFUs/m2, which included
thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea,
vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia.
VNP20009 induced a dose-related increase in the circulation of proinflammatory
cytokines, such as IL-1b, TNF-a,
IL-6, and IL-12. Focal tumor colonization was observed in two patients
receiving 1 x 109 CFUs/m2 and in one patient receiving
3 x 108 CFUs/m2ref.
In all patients treated to date, VNP20009 was not shed in urine or stoolref.
that confers the ability to invade nonprofessional phagocytic cells and
the hly gene from Listeria monocytogenes that allows
expression of lysteriolysin O,
a perforin cytolysin able to perfore phagosomal membranes. This bacterial
vector invades and transfers functional DNA to epithelial cells in vitro.
This strain may be loaded with
locally injected for carcinoma in situ of the bladder expressing
or TNF-a
in TILs
: when reinjected in bloodstream they spontaneously tend to migrate into
the tumor mass)
and CD154
by lentiviral vectors was necessary to significantly improve the APC function
of human autologous primary multiple
myeloma (MM)
cells. Simultaneous CD80/CD154 expression on MM cells allowed the generation
of CD8+ T cells that recognized unmodified MM cells in 11 of
16 cases, specifically in 6 of 6 patients with low-stage disease, but only
in 5 of 10 patients with advanced disease. The activity of CD8+
T cells was MHC restricted and MM specific. In 7 of 7 cases, CD8+
T cell activity was inhibited by mAbs against HLA class I, and in 4 of
4 cases, CD8+ T cells recognized autologous MM cells but not
autologous normal B and T lymphocytes nor bone marrow stromal cells. In
addition, the activity of CD8+ T cells was directed against
allogeneic MM cells that shared at least one MHC allele with the autologous
counterpart, but not against MHC mismatched MM cells. These data lay the
ground for the isolation of new MM antigens and for the design of vaccination
protocols with primary MM cells genetically engineered to express immunostimulatory
moleculesref.,
IL-4
and IFN-g
in BMDCs)
joined to a signaling chain (g, z,
...) capable of initiating T cell activation (ie cytokine release and target
cell lysis).
retroviral vector was engineered that efficiently delivers genes for both
a
and b chains of
TcR
to HSCs
for antigen-specific adoptive immunotherapy. When modified cell
populations were used to reconstruct the hematopoietic lineages of recipient
mice, significant percentages of antigen-specific CD8+ or CD4+
T cells were observed. These cells expressed normal surface markers and
responded to peptide antigen stimulation by proliferation and cytokine
production. Moreover, they could mature into memory cells after peptide
stimulation. Using TcRs specific for a model tumor antigen, the recipient
mice were able to partially resist a challenge with tumor cells carrying
the antigen. By combining cells modified with CD8+- and CD4+-specific
TcRs, and boosting with DCs pulsed with cognate peptides, complete suppression
of tumor could be achieved and even tumors that had become established
would regress and be eliminated after dendritic cell/peptide immunization.
This methodology of "instructive immunotherapy" could be developed for
controlling the growth of human tumors and attacking established pathogensref.
phenotype in favour of the central
memory
subpopulation.
specific for antigens of the targeted cells
and IL-4
in fibroblasts) or chemokines : they can provoke nonspecific inflammatory
cascade.
on vascular endothelium
antisense
: INGN 241, a nonreplicating adenovectorref
: a low dose of live myeloma FO cells induced a cellular immunity against
tumor without additional modulating factors. Lyophilized myeloma FO cells
were used to induce anti-tumor immunity. In a myeloma vaccination model,
immunization with lyophilized myeloma FO cells alone induced a slight response.
However, the immunity was dramatically enhanced by myeloma FO cells transfected
with a recombinant adenovirus, Adv-1/GM-CSF. The immunocytochemical staining
of Adv-1/GM-CSF transfected myeloma FO cells confirmed that > 90% of cells
were positive with GM-CSF expression. Results of sandwich ELISA showed
the amount of secreted GM-CSF was 240 ng/24 h per 106 cells.
Immunization with lyophilized myeloma FO cells secreting GM-CSF prevented
the tumor growth in 60% of BALB/c mice. Antibodies against myeloma FO cells
were found in the sera of immunized mice. Tumor-specific T-cell response
was also evaluated using cytotoxic T lymphocyte assay. In conclusion, lyophilized
myeloma FO cells secreting GM-CSF can be used as a potent vaccine to induce
strong and protective anti-tumor immunityref
with ionizing
radiation (IR)-responsive promoter
(TNFerade® from GenVec Inc.)-specific
antibody : melanomaref
(CD4 T-cell response augmented)
(mbGM-CSF)
: breast carcinomaref
(CD8/CD4 T-cell response augmented ?) and mastocytomaref
(CD8 and CD4 T-cell response augmented)
: sarcomaref
(CD8/CD4 T-cell response augmented ?), mastocytomaref
(CD8 T-cell response augmented) and adenocarcinomaref
(CD8/CD4 T-cell response augmented ?)
: colon carcinomaref
(CD8 and CD4 T-cell response augmented)
: sarcoma,
mastocytomaref,
lymphomaref
and squamous cell carcinomaref
(CD8 T-cell response augmented))
: it is expecially effective against dominant negative mutations (e.g.
X-SCID)
and genes whose expression requires strict control by thier normal promoters
(e.g. HIGM1).
Therapeutic gene delivery typically involves the addition of a transgene
expression cassette to mutant cells. This approach is complicated by transgene
silencing, aberrant transcriptional regulation and insertional mutagenesis.
An alternative strategy is to correct mutations through homologous recombination,
allowing for normal regulation of gene expression from the endogenous locus.
Adeno-associated virus (AAV) vectors containing single-stranded DNA efficiently
transduce cells in vivo and have been shown to target homologous chromosomal
sequences in cultured cells1. To determine whether AAV-mediated gene targeting
can occur in vivo, we developed a mouse model that contains a mutant, nuclear-localized
lacZ gene inserted at the ubiquitously expressed ROSA26 locus. Foci of
-galactosidase-positive hepatocytes were observed in these mice after injection
with an AAV vector containing a lacZ gene fragment, and precise correction
of the 4-bp deletion was demonstrated by gene sequencingref
: it is expecially effective against dominant negative alleles, mutations
which cause inappropriate expression patterns and/or acquisition of new
functions (e.g. oncogenes), genes of infectious agents.
E2 protein is able to regulate negatively the expression of E6 and E7 papilloma
oncoproteins in cervical carcinoma.intrakine
for CCR5
: an intrakine is a modified cytokine with the KDEL ER retention signal
that prevents secretion. The intrakine binds to the newly synthetised CCR5
molecules and traps them in the ER, where they are degraded. RANTES-intrakine
expressing primary CD4+ T lymphocytes were found to resist M-tropic
HIV-1
infection, while maintaining basal biological features).
gp120)
(in b-thalassemia
and SCA)
in CF.
dephosphorylates eIF2a
and inhibits the shutoff of protein synthesis in metabolically-stressed
host cells
inhibits the activation of caspases 1
and 8
and prevents IL-1b
and IL-18
processing.
homolog of Bcl-2
which lacks a loop domain and has poorly conserved BH3
and BH4
domains, escaping negative regulation by pro-apoptotic kinases and caspases.
and paclitaxel
and trimetrexate.
may have some advantages in combating well-established tumors vis-a-vis
either modality administered as a monotherapyref
: in the future, the potential use of stem
cell-derived
cell platforms with gene transfer might allow for a maximally beneficial
approach to regenerative medicineref.
In the stem cell context, one must consider that autologous. stem cells
may be prone to recurrence of the underlying disease that led to end-organ
damage originally. Meanwhile, the use of allogeneic stem cells will entail
immune suppression just as it does in solid organ transplantation. Only
through gene-modification of autologous stem cells can the full potential
of regenerative medicine be realized.
(18 trials have been carried out at October 2002) :
into airway epithelial cells. Transfection efficiency could be increased
through pre-treatment with mucolytics, recombinant DNase or the anticholinergic
drug glycopyrolate. Receptors for Ad and AAV2
are located on the basolateral membrane of human airway epithelial cells
: anyway the transient opening of tight junctions (by EGTA, anti-E-cadherin
antibody, sodium caprate, a blend of sucrose, mannitol and Pluronic F68
or perfluorochemical) is clinically unapplicable given that the airways
of most CF patients are heavily colonised with bacteria,
AAV5
and Sendai
virus (SeV)
and AAV2
vectors can be retargeted to apical surface receptors (B2,
uPAR, P2Y2)
vector expressing CFTR (tgAAVCF) were safe and well tolerated, and resulted
in encouraging trends in improvement in FEV1 and reduction in
inflammation (IL-8) in the sputum of patients with mild CF (FEV1
at least 60% predicted). All subjects receiving tgAAVCF exhibited an increase
(by at least 4-fold) in serum AAV2-neutralizing antibodies and detectable
levels in BAL fluid from 5 of 6 treated subjects undergoing BAL. Gene transfer
but not gene expression was detected in a subset of 6 tgAAVCF subjects
who underwent bronchoscopyref)
can be pseudotyped with glycoproteins from viruses that naturally infect
the airway epithelial cells, such as human
coronavirus 229E,
influenzavirus
A
strains and members of the Filoviridae
family
: systemically transfected hepatocytes with Ad
vectors
:
: the risk of an immune response to the coagulation factor IX (F.IX) transgene
product is a concern. In order to investigate the mechanism of F.IX-specific
lymphocyte activation in the context of AAV gene transfer to skeletal muscle,
AAV-2 vector expressing human F.IX (hF.IX) was injected into outbred immune-competent
mice. Systemic hF.IX levels were transiently detected in the circulation,
but diminished concomitant with activation of CD4+ T and B cells.
ELISPOT assays documented robust responses to hF.IX in the draining lymph
nodes of injected muscle by day 14. Formation of inhibitory antibodies
to hF.IX was observed over a wide range of vector doses, with increased
doses causing stronger immune responses. A prolonged inflammatory reaction
in muscle started at 1.5-2 months, but ultimately failed to eliminate transgene
expression. By 1.5 months, hF.IX antigen re-emerged in circulation in 70%
of animals injected with high vector dose. Hepatic gene transfer elicited
only infrequent and weaker immune responses, with higher vector doses
causing a reduction in T-cell responses to hF.IX. In summary, the data
document substantial influence of target tissue, local antigen presentation,
and antigen levels on lymphocyte responses to F.IXref.
representing a human F.IX-specific CD4+ T cell epitope in hemophilia
B mice. C3H/HeJ mice with a F.IX gene deletion produced inhibitory IgG
to human F.IX after hepatic gene transfer with an AAV vector. These animals
subsequently lost systemic F.IX expression. In contrast, repeated intranasal
administration of the specific peptide resulted in reduced inhibitor formation,
sustained circulating F.IX levels, and sustained partial correction of
coagulation following hepatic gene transfer. This was achieved through
immune deviation to a Th cell response with increased IL-10
and TGF-b production and activation of regulatory
CD4+CD25+ T cellsref
: b-globin
:
: G6PD
: ferrochelatase, others
: spectrin
b,
erythrocytic
: as constitutive expression of CD40L induces lymphomas, the mutation needs
to be corrected while preserving the natural regulation of CD40L using
pre-mRNA trans-splicing (spliceosome-mediated
trans-splicing
(SMaRT)).
Bone marrow from mice lacking CD40L was modified with a lentivirus trans-splicer
encoding the normal CD40L exons 2–5 and was administered to syngenic CD40L-knockout
mice. Recipient mice had corrected CD40L mRNA, antigen-specific IgG1
responses to KLH immunization, regulated CD4+ T-cell CD40L expression
after CD3 stimulation in primary and secondary transplanted mice, attenuation
of Pneumocystis carinii pneumonia, and no evidence of lymphoproliferative
disease over 1 yearref
: WASp
in HSCs.
: T-cells are isolated from blood and stimulated to proliferate in vitro,
before being transduced with a retroviral vector containing the ADA gene.
The cells are then culture-expanded and re-infused into the patients. The
first approved human gene therapy trial was led by Dr William
French Anderson, R. Michael Blaese, and Kenneth Culver in September
1990 at the University of Southern California on a 4-year-old girl, Ashanthi
de Silva. The girl continues to do well but it is unclear whether that
is due to the therapy because she is also on a low-dose regimen of intravenous
synthetic
ADA
therapy, her immune system is now fully functional, with 20% to 25% of
all her T cells containing the gene that was introduced by retroviral transferref
: CD132
/ IL-2Rgc-mediated
expression of gc in ex vivo-transfected
HSCs of 11 X-linked SCID patients from 5 countries (successful in 9 patients),
was suspended in October 2002 as in a 3-year-old boy retroviral integration
inside LMO2
oncosuppressor gene (which encodes a transcription factor required for
hematopoiesis) had caused g/d
T cell leukemia, first apparent in August, 30 months after the gene therapy.
Another boy fell ill on January 2003 due to integration near enough to
LMO2 gene, too. These patients were the youngest to be treated—just 1 and
3 months—while the others were all at least 6 months old. They also received
the highest number of corrected cells : so, statistically speaking, the
more you inject, the higher the probability. Anyway gene therapy has a
relatively good risk–benefit ratio : all 14 children treated to date are
alive and well, and the 2 boys who developed leukemia responded well to
chemotherapy and are in complete clinical remissionref.
A third, 3-years old, boy developed leukemia and died in February 2005
: FDA suspended trials in USA. 7 children and 1 adult have been treated
at Great Ormond Street Hospital in London.
: gp91phox, p47phox
: CD18
: ALDP
: arylsulfatase
A (ARSA) / cerebroside sulfatase
: acid b-glucosidase
/b-glucocerebrosidase
/ glucosylceramidase
: a-L-iduronidase.
might be treated by transferring the gene encoding for the LDL-R
into hepatocytes in order to reduce the level of serum cholesterol.
: IGF-1
targeting to rat muscles induces growth in size and strength by 15 to 30%
(when they also put through an exercise program, the animals double their
muscle strength).
: most effort to replace the defective dystrophin
gene has concerned an ex vivo strategy to transfect isolated muscle
cells with multiple copies of the dystrophin gene and re-implant the cells
back into the patient’s muscle. There has so far been little work on the
in
vivo delivery of the dystrophin gene as there are few vectors suitable
for the efficient delivery of genes into muscle cells in vivo and
ensure tissue specific expression, although AAV
can deliver microdystrophinref.
Systemic gene delivery into muscle has been a major challenge for muscular
dystrophy gene therapy, with capillary blood vessels posing the principle
barrier and limiting vector dissemination. Previous efforts to deliver
genes into multiple muscles have relied on isolated vessel perfusion or
pharmacological interventions to enforce broad vector distribution. The
efficiency of multiple AAV vectors was compared after a single injection
via intraperitoneal or intravenous routes without additional intervention.
AAV8 is the most efficient vector for crossing the blood vessel barrier
to attain systemic gene transfer in both skeletal and cardiac muscles of
mice and hamsters. Serotypes such as AAV1 and AAV6, which demonstrate robust
infection in skeletal muscle cells, were less effective in crossing the
blood vessel barrier. Gene expression persisted in muscle and heart, but
diminished in tissues undergoing rapid cell division, such as neonatal
liverref.
: LARGE
glycosyltransferase overexpression can functionally bypass a-dystroglycan
(a-DG) hypoglycosylation and marked reduction
of its receptor function in Largemyd miceref:
direct injection of naked DNA coding for transglutaminase
1 (TGM1) in keratinocytes.
: injection of naked DNA coding for laminin
b3
in keratinocytes.:
helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding
sequences, induce prolonged transgene expression and exhibit significantly
less chronic toxicity than early-generation Ad vectors. A HD-Ad vector
has been used to achieve liver-restricted expression of human uridine diphospho-glucuronosyl
transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma
bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for
>2 yr after a single i.v. administration of vector expressing the therapeutic
transgene at a dose of 3 x 1012 viral particles per kg. HPLC
analysis of bile from treated rats showed the presence of bilirubin glucuronides
at normal WT levels >2 yr after 1 injection of vector, and i.v. injection
of bilirubins IIIa and XIIIa
in the same animals revealed excess bilirubin-conjugating capacity. There
was no significant elevation of liver enzymes (alanine aminotransferase)
and only transient, moderate thrombocytopenia after injection of the vector.
A clinically significant reduction in serum bilirubin was observed with
a dose as low as 6 x 1011 viral particles per kg. Complete,
long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar
syndrome can be achieved with one injection of HD-Ad vector and negligible
chronic toxicityref.