Download NOD/SCID mice

Jianfei Xie
2015.07.19
Introduction
Hemophilia A is an X-linked recessive bleeding disorder
(Morvarid Moayeri et al. 2004).
Introduction
Hemophilia A is characterized by inability to clot blood because
of FVIII gene mutations and deficiency of this coagulation factor
(Follenzi et al. 2012).
3D structure of FVIII, PDB ID: 2R7E
FVIII deficiency results in a severe bleeding phenotype leading
to death, significant illness (Yi Lin et al. 2002).
Joint injury
Severe bleeding
Hemophilia A affects one in 5000 males, or about 400,000
individuals worldwide (Mannucci et al. 2014).
FVIII injection
Currently, Hemophilia A is treated by administration of plasma
derived or recombinant FVIII, but this strategy is complicated
by the development of inhibitory antibodies (Diego Zanolini et
al. 2015). These so-called inhibitors may jeopardize the
patient’s life and make therapeutic management more
complex, and costly(Thierry Calvez et al. 2014).
Inhibitors
FVIII Modification
[1] Recombinant canine B-domain deleted FVIII exhibits high specific
activity and is safe in the canine hemophilia A model. Denise et al. Blood.
2009.
[2] Minimal modification in the factor VIII B-domain sequence ameliorates
the murine hemophilia A phenotype. Joshua et al. Blood. 2013.
[3] Reduction of the inhibitory antibody response to human factor VIII in
hemophilia A mice by mutagenesis of the A2 domain B-cell epitope. Ernest
et al. Blood. 2004
[4] Noncovalent stabilization of the factor VIII A2 domain enhances efficacy
in hemophilia A mouse vascular injury models.Lilley Leong et al. Blood.
2014.
Gene therapy
Naked DNA transfer of Factor VIII induced transgene-specific,
species-independent immune response in hemophilia A mice.
Mol Ther. Ye et al. 2004.
FVIII disappearance correlated with the generation of high-titer,
inhibitory anti-FVIII antibodies.
Inhibitors
Cell therapy
The goal of cell therapy is to introduce long-term expression
of therapeutic levels of FVIII in vivo resulting in a cure of the
disease .
Where is factor VIII synthesized?
Controversial !
Hepatic and Extrahepatic
Hepatic
1. Liver sinusoidal endothelial cells (LSEC): Main source
(Everett LA et al, 2014; Fahs SA et al, 2014; Diego Zanolini et al, 2015)
2. Hepatocytes and Küpffer cells: Low level
(Diego Zanolini et al 2015)
Extrahepatic
1. Peripheral or cord blood derived cells, including:
(1) Endothelial progenitor/endothelial cells.
(2) Monocytes, macrophages and megakaryocytes from
hematopoietic stem cells.
2. Bone marrow derived cells, including
Monocytes, macrophages and endothelial cells.
These extrahepatic cells could synthesize factor VIII in sufficient
amount to ameliorate the bleeding phenotype in hemophilic
mice (Diego Zanolini et al 2015)
Cell therapy protocols:
Hepatic and Extrahepatic
Therapy Model:
1. Mouse to mouse
2. Human to mouse
3. Human to human
Mouse to mouse
[1] Transplanted endothelial cells repopulate the liver endothelium and correct
the phenotype of hemophilia A mice. Antonia Follenzi et al, 2008.
[2] Transplantation of endothelial cells corrects the phenotype in hemophilia A
mice. V. Kumaran et al. 2005.
[3] Factor VIII Can Be Synthesized in Hemophilia A Mice Liver by Bone
Marrow Progenitor Cell-Derived Hepatocytes and Sinusoidal Endothelial Cells.
Neelam Yadav. 2012.
[4] Role of bone marrow transplantation for correcting hemophilia A in mice.
Antonia Follenzi. Thrombosis and hemostasis. 2012.
[5] The therapeutic effect of bone marrow–derived liver cells in the phenotypic
correction of murine hemophilia A. Neelam Yadav. Thrombosis and
hemostasis. 2009.
[6] Phenotypic correction of murine hemophilia A using an IPS cell-based
therapy. Dan Xu. 2009
[7] Sustained phenotypic correction of hemophilia a mice following
oncoretroviral-mediated expression of a bioengineered human factor VIII
gene in long-term hematopoietic repopulating cells. The Journal of Gene
Medicine. Jiro Kikuchi. 2004.
Human to mouse
Hepatic
[1] Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells
Transplanted in Immunodeficient uPA Mice. Marina E. Fomin. 2013.
[2] Human hepatocyte propagation system in the mouse livers functional
maintenance of the production of coagulation and anticoagulation factors.
Cell Transplantation. Kohei Tatsumi et al. 2012.
uPA/SCID mice model
This mice model have a feature to develop an active damage
of their own hepatic parenchymal cell, provide a hepatic
environment that is more conductive to the engraftment and
proliferation of human cells (Tateno et al. 2004; Kohei
Tatsumi et al. 2012).
Human to mouse
Extrahepatic (4 papers)
1. Use of blood outgrowth endothelial cells for gene therapy
for hemophilia A. Yi Lin et al. Blood. 2002
Human venous blood  Mononuclear  Endothelial cells
 Transduced with GFP-FVIII expression vector
 NOD/SCID mice model
Yi Lin et al. Blood. 2002
Yi Lin et al
Blood. 2002
Human to mouse
Extrahepatic
2. Sustained transgene expression by human cord blood
derived CD34+ cells transduced with simian immunodeficiency
virus agmTYO1-based vectors. Kikuchi et al. J Gene Med. 2004.
NOD/SCID mice
Human cord blood  CD34+ cells
 Transduced with GFP-FVIII expression vector
 NOD/SCID mice model
Presence of human CD 45+ cells
Kikuchi et al. J Gene Med. 2004
Plasma FVIII levels in transplanted NOD/SCID mice
Kikuchi et al. J Gene Med. 2004
Expression of lineage markers in human CD45+ cells and detection of
human CD41+ platelets . Kikuchi et al. J Gene Med. 2004.
Human to mouse
Extrahepatic
3. Extrahepatic sources of factor VIII potentially contribute to
the coagulation cascade correcting the bleeding phenotype of
mice with hemophilia A. Diego Zanolini. Haematologica. 2015
Human cord blood  CD34+ cells
 NOD/SCID-γNull Hemophilia A mice (NSG-HA) model
Superior for transplanting human cells (Ito M et al, 2002)
Diego Zanolini. Haematologica. 2015
Diego Zanolini. Haematologica. 2015
Expression of FVIII in human Liver sinusoidal endothelial cells
Diego Zanolini. Haematologica. 2015
Quantitative PCR showing FVIII mRNA expression in human
liver and isolated human LSEC, KC and hepatocytes
Diego Zanolini. Haematologica. 2015
Diego Zanolini. Haematologica. 2015
Transplanted human cells were identified in mouse blood by
cytofluorimetry for human CD45 marker
Diego Zanolini. Haematologica. 2015.
Human chimerism in BM and spleen of transplanted mice
Diego Zanolini. Haematologica. 2015.
FVIII activity in mice transplanted with human CD34+ cells
Diego Zanolini. Haematologica. 2015.
Three months after transplantation mice were challenged
with tail clipping and nine out of 12 (75%) survived
Diego Zanolini. Haematologica. 2015.
Human to mouse
Extrahepatic
4. Platelet gene therapy corrects the hemophilic phenotype in
immunocompromised hemophilia A mice transplanted with
genetically manipulated human cord blood stem cells. Qizhen
Shi. Blood. 2014.
Human cord blood  CD34+ cells
 Transduced with 2bF8LV vector
FVIII expression is under the control of the platelet-specific
glycoprotein IIb promoter (2bF8).
 NOD/SCID-γNull Hemophilia A mice (NSG-HA) model
Human cell chimerism in mice
Qizhen Shi. Blood. 2014.
Human cell chimerism in mice
Qizhen Shi. Blood. 2014.
Analysis of platelet FVIII expression in mice
Qizhen Shi. Blood. 2014.
Survival rate after tail chipping
Qizhen Shi. Blood. 2014.
Human to human
[1] Cord blood hematopoietic stem cell transplantation in an
adolescent with haemophilia. Caselli D. Haemophilia.
2012;18(2): 48-49.
The authors described that during follow-up the FVIII levels in the plasma
of this patient were increased and that, on the basis of the international
classification, the degree of his coagulation defect prior to transplantation
qualified him as a severe case whereas after transplant he should be
defined as having moderate hemophilia.
[2] Allogeneic bone marrow transplantation in a child with
severe aplastic anemia and hemophilia A. Ostronoff M. Bone
Marrow Transplant. 2006;37(6):627-628.
The FVIII level did not change after transplantation, suggesting that bone
marrow does not contribute significantly to FVIII production
Our Research
Preclinical Therapy of Expanded and Differentiated Endothelial
Progenitor Cells from Human and Non-human Primates
Related articles:
[1] Sustained Expansion and Transgene Expression of Coagulation Factor
VIII-Transduced Cord Blood-Derived Endothelial Progenitor Cells. Christian
Herder et al. Arterioscler Thromb Vasc Biol. 2003.
[2] Storage and regulated secretion of factor VIII in blood outgrowth
endothelial cells Biggelaar et al. Haematologica 2009.
(1) In these two research human cells were transduced with a
lentivirus encoding FVIII-GFP.
(2) These two papers did not have mouse model.
Next key content
1. Human FVIII detection in mice.
2. Survival rate after tail chipping.
My question
Can Endothelial Progenitor/Endothelial Cells transplant in
liver without the Liver sinusoidal endothelial cells injury step?
Protocol
1. Obtain immunodeficient hemophilia A mice
2. Immunosuppression (immunosuppressive reagent or T cells)
[1] Immunomodulation of transgene responses following naked DNA transfer
of human factor VIII into hemophilia A mice. Carol H. Miao. Blood 2006.
[2] Donor antigen-primed regulatory T cells permit liver regeneration and
phenotype correction in hemophilia A mouse by allogeneic bone marrow
stem cells. Kochat V. Stem Cell Res Ther. 2015
3. Encapsulation
[1] Encapsulated human primary myoblasts deliver functional hFIX in
hemophilic mice. J Gene Med. Jianping Wen. 2007.
[2] Delivery of human factor IX in mice by encapsulated recombinant
myoblasts a novel approach towards allogeneic gene therapy of
hemophilia B. Hortelano et al. Blood. 1996.