in utero hematopoietic stem cell transplantation in ... · canine model we have developed a model...
TRANSCRIPT
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IN UTERO HEMATOPOIETIC STEM CELL TRANSPLANTATION IN CANINES: THE GESTATIONAL
WINDOW OF OPPORTUNITY TO MAXIMIZE ENGRAFTMENT
Karin J. Blakemore, M.D.
Division of Maternal-Fetal Medicine The Bone Marrow Transplant Program
The Johns Hopkins University School Of Medicine
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Introduction
In Utero Hematopoietic Stem Cell Transplantation (IUHSCT) is a promising therapy that may circumvent many of the morbidities and limitations of postnatal BMT.
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Introduction
Candidate diseases for human application of IUHSCT include –Hemoglobinopathies –Granulocyte disorders –Leukodystrophies –Storage disorders
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Theoretical Benefits of IUHSCT
Avoid ablative therapy required for donor cell expansion in postnatal HSCT
GVHD is less likely to occur
Avoid the need for a HLA-matched donor
Low-level engraftment may be curative or allow same-donor postnatal HSCT by induction of host tolerance
Avoid organ damage if performed early enough
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Previous IUHSCT in Humans
The only clear successes to date have been with immunodeficiencies (X-linked SCID)
The major problem has been lack of engraftment
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In Utero Bone Marrow Transplantation
Clinical Pre-Clinical
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Our overall objective is to increase our understanding of the parameters involved in safe and successful in utero BMT in an animal model which will be relevant to the human.
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Our long term goal is to establish a therapeutic protocol to cure congenital hemoglobinopathies and metabolic disorders such as congenital leukodystrophies.
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Canine Model
We have developed a model of IUHSCT in
fetal canines which, similar to humans,
develop immunocompetency prior to birth,
in order to investigate the roles of donor cell
dosing and gestational age in engraftment
and GVHD.
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Benefits of Canine Model
Relatively short gestational length compared to other animal models
Litter size allows multiple experiments to be performed within the same gestation
Uterus is tolerant of surgical manipulation and few obstetrical complications
Canines have many analogous medical conditions for future treatment trials
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Benefits of Canine Model
Availability of:
– Canine DNA markers for engraftment analyses
– Canine CD34 antibody
Hematopoietic stem cells
– Canine CD3 and CD5 antibodies
T cells
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Background
The development of the canine fetal immune system shares similarities with humans that are relevant to a preclinical model of IUHSCT.
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Canine Immune Development
Unlike rodents, dogs appear to be immunologically mature at birth.
The canine fetus exhibits bacteriophage recognition at 40 gestational days (term day 63).
Peripheralization of lymphocytes begins to occur between day 45-50.
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Canine Immune Development
Thymectomy of the 48 day gestational dog produces deficient humoral antibody and cellular hypersensitivity response.
The earliest detectable antibody production occurs at ~day 56.
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Background
Previous work by others have proposed a gestational age “window of opportunity” which permits successful engraftment and avoidance of GVHD following IUHSCT.
a). Early enough to avoid graft rejection
b). Late enough to minimize competition from highly proliferative fetal host cells
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The Concept of a Window of Opportunity
a). The “window of opportunity” for IUHSCT should precede the establishment of host immunologic competence.
b). IUHSCT too early might be less successful during the rapid cellular expansion of fetal hematopoietic cells.
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Objective
To investigate the optimal time in gestation for IUHSCT in a canine model.
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Study Design
Eight bred animal pairs were procured for IUHSCT at post-fertilization day 31-50.
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Study design: Donor HSC Preparation
Paternal canine bone marrow harvested
Mononuclear cells isolated – CD34+ selection with Miltenyi magnetic bead
system (1H6 clone)
or
– Entire ficolled fraction used
Flow cytometry for CD34 cell purity (2E9 clone)
Flow cytometry for CD3/CD5 percentages
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Study design: Donor T cell Preparation
Paternal canine peripheral blood harvested
Mononuclear cells isolated
CD3+ selection with Miltenyi magnetic bead system
Flow cytometry to determine cell purity using CD3 and CD5 antibodies
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Study design: In Utero HSCT procedure
Sonography to verify gestational age
General anesthesia
Laparotomy to expose the uterus
Intraperitoneal fetal injection under ultrasound guidance
– HSCs and T cells (recipients)
– Ethiodol dye (controls)
Close abdomen after checking fetal viability
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Recipient Control
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Study design: Postnatal Studies for Engraftment
DNA Engraftment Analyses
– TaqMan canine SRY for female recipients
– Unique microsatellite loci using capillary electrophoresis for both sexes
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Study design:
Tissues examined
Blood
Bone Marrow
Liver
Spleen
Thymus
Brain
Kidney
Heart
Skin
Large Intestine
Tongue
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Results
Days
@Tx
CD34(10E9)
cells/kg
T-cell (10E9)
cells/kg
Max Engraftment
Blood/BM %
31 3.2 1.1 0
32 3.4 1.1 0.53
36 2.5 0.63 FDIU @ IUHSCT
38 1.1 0.65 3
42 0.35 0.33 7.5
46 0.09 0.11 5
47 0.09 0.14 1.4
50 0.24 0.19 0.51
Term survival 77% (36/47); total of 22 recipient animals.
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0
2
4
6
8
10
12
30 35 40 45 50
Gestational Day at IUHSCT
% E
ngra
ftm
ent
hem
ato
poie
tic tis
sues
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0
1
2
3
4
5
6
7
8
31 32 36 38 42 46 47 50
Days gestation at transplant
En
graft
men
t
Engraft-Blood
Engraft-BM
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0
2
4
6
8
10
12
30 35 40 45 50
Gestational Day at IUHSCT
% E
ngra
ftm
ent
hem
ato
poie
tic tis
sues
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0
2
4
6
8
10
12
31 32 36 38 42 46 47 50
Days gestation at transplant
En
graft
men
t Engraft-Blood
Engraft-BM
Engraft-Spleen
Engraft Liver
Engraft-Thym
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Conclusions
Fetal age at time of injection of paternally derived HSCs may play a key role in achieving engraftment in our canine model of IUHSCT.
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Conclusions
A gestational age window at ~42 days may permit maximal engraftment opportunity without GVHD in the fetal canine.
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Our work supports the concept that
At early gestational ages, rapid proliferation of fetal hematopoietic cells may prohibit engraftment.
At later gestational ages, maturation of the fetal immunologic response may lead to graft rejection.
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A window in gestation in which host cellular proliferation is slowing yet spaces for donor cells to occupy are still available may prescribe the optimal time to perform IUHSCT.
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Future Directions
Future studies will focus on cell dosaging within the gestational age window of 39-45 days to determine parameters for maximal engraftment in this mammalian model of IUHSCT.
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Future Directions
Incremental adjustments in CD34+ and T cell dosages.
Long term engraftment studies.
Investigate parameters that achieve GVHD in the canine model.
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Future Directions
Test the therapeutic potential of engraftment achieved through IUHSCT in canine models of human disorders.
Studies in animal models such as the canine will provide a framework for advancing IUHSCT towards human application.
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In Utero Bone Marrow Transplantation
Clinical Pre-Clinical
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Co-Investigators:
Scott Petersen, M.D., Mariya Gendelman, BA, Kathleen Murphy PhD,
Chris Bird, David Hummel, BS,
Michael Tobenson, M.D., Gail Stetten PhD,
Richard Jones, M.D.