Cancer Projects
Project:
Targeted Liposomes
as a Novel Delivery Method for
Nucleic Acids.
Jeffrey
Hughes, Ph.D. and Nancy
Cheng, Ph.D. (Burroughs Welcome
Foundation)
Non-viral vectors
will
be developed for the selective
delivery of genes to malignant
cells. The vector will exploit
the increased requirement of
rapidly growing cells for more
nutrients
by attaching a nutrient-ligand
onto
the vector (liposome). The vector
additionally will have a positively
charged lipid to enhance nucleic
acid binding along with a novel
pH sensitive surfactant. The
role of the surfactant is to
increase the amount of nucleic
acid
escaping the endosome and correspondingly
increase
the transfection efficiency.
The vector system will be evaluated
first
in an animal model of cancer.
Preliminary
studies will be carried out using
a marker gene (beta-galactosidase)
with
later experiments using a gene
encoding for cytosine deaminase.
Cytosine
deaminase can catalyze the conversion
of the innocuous agent 5-fluoro
cytosine to the anti-cancer agent
5-fluorouracil. By selective
delivery
of this gene to only cancer cells
the therapeutic index of 5-fluorouracil
can then be increased.
In
this proposal the development
of a soft pH sensitive surfactant
will be evaluated. This agent
will become active at endosomal
pH, has membrane disrupting effects,
and will be inactivated before reaching the lysosome. For this
agent to work, the surfactant
must enter the cell by endocytosis.
To accomplish this, the soft
surfactant will be incorporated
into liposomes that have been
shown to be enter cells in this
manner. The novelty of this delivery
system stems from the soft pH
sensitive surfactant (SPS). The
synthetic features of this route
include a pH sensitive region
(imidazole), the lipophilic moiety
(dodecanol), and the enzymatically
cleavable connector (2-bromopropionyl bromide). The SPS will
be incorporated into a liposomal
nucleic acid carrier with other
lipid components. A spacer (polyethylene
glycol 3,000) will be attached
between the ligand and liposome
to increase natural ligand: receptor
interactions and a positively
charged metabolically cleavable
cationic lipid 1,2, dioleoyl-3-trimethylammonium-propane.
Liposomes will be prepared in
the HAL facility by hydration
of a dried lipid layer. After
the liposomes are formed, they
will be extruded through a polycarbonate
membrane to a final size of 100
nm. The N-hydroxylsuccinamide
esters of the ligands will be
reacted with the performed liposomes.
Free ligand will be removed by
gel chromatography. The experimental
approach will identify various
receptor mediated pathways that
will be optimal for delivery of genes to malignant cells. Once
the best delivery system is chosen,
animal studies will be conducted
with a plasmid encoding for cytosine
deaminase and, if successful,
the progression into human studies
on the GCRC could proceed rapidly,
probably within 4 years.
Reference
- Hughes JA, Avrotskaya Av, Juliano RL. Oligonucleotide transport
across membranes into cells: effects
of chemical modifications. In, Delivery Systems for Antisense
oligonucleotide therapeutics. Oxford, CRC Press, 1994.
Project:
Drug Resistance of Normal Hematopoietic Stem Cells.
Jan
Moreb, M.D., and James
R. Zucali, Ph.D.
We have previously shown that preincubation with interleukin-1 (IL-1) and
tumor necrosis factor alpha (TNFa) can protect normal hematopoietic progenitors
but not leukemic cells from the toxicity of 4-hydroperoxy-cyclophosphamide
(4-HC), an active derivative of cyclophosphamide. Diethylamino- benzaldehyde
(DEAB), which inhibits aldehyde dehydrogenase class 1 (ALDH-1), the enzyme
responsible for inactivation of 4-HC, abolishes this protection. Thus,
the general goal of this proposal is to study the role of IL-1, TNFa, and
ALDH in the protection of normal and tumor cells from 4-HC. Northern and
Western analysis show induction of ALDH-1 in mRNA and protein in human
bone marrow cells, with proportional two-fold increase in the ALDH-1 activity
after incubation with IL-1 and TNFa. The full length of ALDH-1 cDNA was
synthesized and subcloned in pLNCX retroviral vectors in the sense and
antisense orientation. The expression of ALDH-1 in the sense or antisense
orientations in appropriate cell lines will determine the relationship
between ALDH-1 and resistance to 4-HC. Another specific aim in this proposal
is to determine the effect of overexpression of ALDH-1 in human normal
hematopoietic progenitors on their in vitro resistance to 4-HC using colony
forming assay and long-term bone marrow cultures. These studies will impact
molecular engineering in relation to drug resistance of normal and cancer
cells, as well as gene therapy in general. Although our preliminary studies
are being done with retroviral vectors, we are also exploring the use of
AAV for hematopoietic gene delivery in collaboration with the Vector Core
Laboratory.
This proposal will be the basis for future clinical trials
where normal bone marrow cells will be targeted with such genes,
e.g., aldehyde dehydrogenase and manganese superoxide dismutase (see
the proposal of Dr. Zucali), with the aim to render these cells resistant
to combination chemotherapy and radiotherapy and eliminate the need
for support after high-dose therapy with stem cells. The HAL will
be very critical in terms of providing the vectors for human use.
The human studies will target diseases that can be treated with combination
chemo/radiotherapy, such as hymphoma and multiple myeloma, or other
hematopoietic malignancies, provided that purified normal stem cells
can be obtained for the gene transduction. Clinical studies could
start in 3-5 years. Both inpatient and outpatient GCRC facilities
will be required to accommodate these patients.
Reference
- Zucali JR, Moreb J, Gibbons W, Alderman J, Suresh A, Zhang
Y, Shelby B. Radioprotection of hematopoietic
stem cells by interleukin-1. Exp Hematol 22:130-135, 1994.
Project:
Mechanisms of Radioprotection in Hematopoiesis.
James R. Zucali, Ph.D.
Interleukin-1 and tumor necrosis factor have previously been shown to protect
mice against lethal irradiation. They are also known to induce the expression
of the antioxidant enzyme manganese superoxide dismutase (MnSOD) which
may be responsible in some part for the radioprotection. The goal of this
investigation to determine if up-regulation of MnSOD will confer increased
protection in patients from lethal irradiation for hematopoietic cells
in order to provide a therapeutic advantage in the selective protection
of normal versus malignant cells. To accomplish this, we have shown that
transfection of a melanoma cell line with MnSOD in the sense direction
will provide increased resistance to irradiation and increased MnSOD message
and protein, whereas, transfection of the leukemic cell line K562 with
MnSOD in the antisense orientation demonstrates increased sensitivity to
irradiation and decreased MnSOD message and protein. Studies are currently
underway to transduce normal murine bone marrow stem cells with the gene
for MnSOD using both retroviral and AAV vectors to determine if overexpression
of this enzyme in hematopoietic stem cells will provide increased protection
from an irradiation insult in a lethally irradiated mouse model system.
Success with this system would lead to proposing a clinical investigation
of inserting the MnSOD gene into human hematopoietic stem cells for protection
against irradiation in a bone marrow transplant setting. To accomplish
this would require a GMP vector production laboratory and associated personnel
in the GCRC and the Gene Therapy Center.
References
- Zucali JR. Mechanism of Protection of Hematopoietic
Stem Cells from Irradiation. Leukemia and
Lymphoma 13:27-32, 1993.
- Zucali JR, Moreb J, Gibbons W, Alderman
J, Suresh A, Zhang Y, Shelby B. Radioprotection
of Hematopoietic Stem Cells by Interleukin-1.
Exper. Hematol. 22:130-135, 1994.
- Suresh A, Tung F, Moreb J, Zucali JR. Role of Superoxide Dismutase
in Radioprotection Using
Gene Transfer Studies. Cancer Gene Therapy 1:85-90,
1994.
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