Keywords hepatocytes - expression profiling - methylation profiling - expression–methylation
correlation - endothelial cells - F8 protein - gene therapy
Current Hemophilia Treatment Strategies
Current Hemophilia Treatment Strategies
The factor VIII (FVIII) protein plays an important role in blood coagulation as a
cofactor of FIX to form the tenase complex that activates FX.[1 ] The main cells producing and secreting FVIII are the sinusoidal endothelial cells
of the liver.[2 ]
[3 ]
[4 ] This has been proven in both mice and human cells.[4 ]
[5 ] Deficiency in FVIII leads to hemophilia A; the symptoms involve spontaneous bleeding
in joints, muscles, and other sites. Despite the efficient current prophylaxis regimes
with intravenous FVIII concentrates or subcutaneous FVIII mimetic bispecific antibody,
most patients develop joint arthropathy sooner or later in life.[6 ]
[7 ]
[8 ] Actually, there is no effective cure to FVIII deficiency.
FVIII Protein Synthesis and Regulation
FVIII Protein Synthesis and Regulation
The synthesis and secretion of FVIII molecule in human is highly regulated in time
and space: it takes place in specialized cells. Recently, it has been accepted that
the endothelial cells and not the hepatocytes are the main source of blood-derived
FVIII.[3 ]
[5 ] Additionally, the liver organ is known to be the major contributor to the secreted
FVIII; therefore, the liver sinusoidal endothelial cells (LSECs) are expected to be
the main cells that largely contribute to the FVIII levels.
Wild-type FVIII protein consists of six domains, A1–A2–B–A3–C1–C2, making 2,332 amino
acids, of which the first 19 aa are coding for a signal peptide that guide the intracellular
trafficking of the FVIII protein into the conventional secretary pathway: ER to Golgi
to extracellular space. During this process, the FVIII protein is known to interact
with proteins in the ER–Golgi compartments. Among the known interacting partners are
the three ER lumen proteins: the immunoglobulin-binding protein (BiP or HSPA5),[9 ] Calnexin (CNX), and Calreticulin (CRT).[3 ] These chaperons have the role (as was known) to control the correct folding and
the quality of the F8 protein, and only when it has the correct folding it will be
released from the ER to the Golgi. Two further chaperons (LMAN1 and MCFD2) are known
to interact with FVIII in the endoplasmic reticulum–Golgi intermediate compartment
(ERGIC) and facilitate the transport of FVIII from the endoplasmic reticulum to the
Golgi compartment.[10 ]
[11 ]
[12 ] In addition to the before mentioned proteins, it is expected that additional proteins
will play role in the secretion process. Thus, the intracellular synthesis and secretion
of FVIII protein is highly regulated and requires complex intracellular machinery.[13 ]
Current Gene Therapy Protocols in Hemophilia A
Current Gene Therapy Protocols in Hemophilia A
Current strategies toward a future cure for FVIII deficiency include gene and cellular
therapy. Gene therapy protocols using the AAV as a vector system provide an actual
option that at least could partially cure the disease by substantially increasing
FVIII activity in patients to even normal levels. Indeed current clinical trials showed
promising results.[14 ] However, the AAV vector approach is limited as about 50% of the patient population
has already existing antibodies against the AAV capsid that may cross-react over various
AAV subtypes.[15 ]
[16 ] Rangarajan et al[15 ] reported an almost complete normalization of the mean FVIII activity in the first
year. Pasi and coworkers[16 ] showed that the factor levels over a period of 3 years decline from a median of
60 IU/dL after 1 year to a median of 20 IU/dL after 3 years, but still remained in
a range that protects patients from bleeds. While this clinical study demonstrates
the success of gene therapy as a proof of principle, it also reveals a high range
of FVIII protein expression ranging from quite low levels to supranormal levels.[15 ]
[17 ] Furthermore, about 70% of the patients required immune suppression with corticosteroids—in
some of them for a significant time period. Moreover, it is not clear whether the
FVIII activity will further decline with time or reach a plateau.[16 ] The need for immune suppression over months led to the questions whether there are
intracellular mechanisms contributing to an elevation of liver enzymes as cellular
stress, since the hepatocyte is not the natural site of FVIII protein synthesis. Moreover,
codon optimization may lead to a higher content of unmethylated CpG dinucleotides
and subsequently to a toll-like receptor-9–mediated immune response. In contrast,
an ideal gene therapy should (1) deliver the expressing gene cassette to the specific
cell that naturally secrete FVIII, (2) guarantee a nonharmful integration of the gene
in the cell, (3) sustain of continuous expression, (4) mimic the natural control process
of expression and secretion, and (5) have a natural promoter to ensure natural levels
of expression.
Current FVIII gene therapy protocols rely on AAV delivery vectors and on highly active
non–factor VIII promoters specific for hepatocytes (like Tie2 promoter) that are currently
needed and necessary to overcome the inefficient delivery tools and transfection efficiency.
As a result, the low percentage of transfected cells is overexpressing FVIII that
could lead to high stress level on the positively transfected cells and unnatural
production of FVIII protein (with abnormal posttranslational modifications).
Alternative Cellular Therapy Strategies
Alternative Cellular Therapy Strategies
An alternative to transferring only the FVIII gene, the concept of cellular therapy
is based on use of entire therapeutic cells (i.e., LSECs): Transfer of cells that
are naturally producing FVIII proves to be effective in treating hemophilia conditions,
at least in mice.[18 ]
[19 ]
[20 ] By this, some of the earlier-listed problems associated with gene transfer might
be overcome, mainly the natural control of production where the cells are responding
to incoming signals and produce natural product without stressing the cells.
Most interestingly, in September 2020, a phase I/II clinical trial has been started
by Sigilon Therapeutics implanting encapsulated FVIII spheres by laparoscopy in patients
with hemophilia A. They are using genetically modified cells with a nonviral vector
to produce B-domain–deleted human FVIII (https://clinicaltrials.gov/ct2/show/NCT04541628 ).
In the next section, we present data that highlight the specific molecular signatures
of LSECs in comparison to both hepatocytes and endothelial cells.
Comparison of Fetal LSEC with other Fetal Endothelial Cells
Comparison of Fetal LSEC with other Fetal Endothelial Cells
To investigate if LSEC could be distinguished from other fetal endothelial cells already
at early stage of development, we compared them with other fetal endothelial cells
(Jamil et. al., Molecular Analysis of Fetal and Adult Primary Human Liver Sinusoidal
Endothelial Cells: A Comparison to Other Endothelial Cells, Int. J. Mol. Sci, In press).
Fetal LSECs (f-LSECs) were compared with other fetal endothelial cells (HCMEC, HPAEC,
and HPMEC). A significant number of differentially expressed genes and differentially
methylated CpGs were detected.
Ontology enrichment analysis in LSECs showed grouping of differential expression genes
in categories like positive cell regulation and involved morphogenesis development.
Enrichment in endothelial cells included establishment of protein targeting ([Fig. 1A ]). Moreover, ingenuity pathway analysis (IPA) of disease and biofunction predicted
significant enrichment in biofunctions in LSECs related to cell movement, hematological
system development and function, immune cell trafficking, and inflammatory response.
Enrichment in other endothelial cells included infectious diseases, organismal survival
injury, and abnormalities ([Fig. 1B ]).
Fig. 1 Comparison of LSECs and other endothelial cells. (A ) Significant enriched ontologies of the genes that are showing difference in expression
for biological process. Red and blue circles represent enriched group of genes in
LSECs and ECs, respectively. Empty black circles represent wider ontology group that
includes the smaller filled circles. Blue lines are connecting nodes with common genes.
(B ) Disease and functions identified for differentially expressed genes between LSEC
fetal and other fetal ECs. The upper panel shows a heat map representing the enrichment
of differentially expressed genes between LSECs and other endothelial cells. Gross
categories are represented by gross rectangles with headings (like cellular movement
and cancer), while smaller subcategories are represented by smaller rectangles. Lower
panel lists the top 5 diseases and functions overrepresented in LSECs (orange numbered
boxes in upper panel with positive Z scores in table) and the top 5 diseases and functions overrepresented in other endothelial
cells (blue numbered boxes in upper panel with negative Z scores in table). ECs, endothelial cells; LSECs, liver sinusoidal endothelial cells.
Comparison of LSECs with Hepatocytes
Comparison of LSECs with Hepatocytes
Both LSECs and hepatocytes are main cellular constituents of liver tissue. Although
they could share some gene expression fingerprint as belonging to the same organ,
they are obviously different. To show the differences, we have compared the expression
profile using biological process ontology and the IPA disease and biofunction ([Fig. 2 ]). The biological process gene ontology showed enrichment in hepatocytes in small
metabolic process, toxic substance detoxification, innate immune response, negative
activity of peptidase, and others—the main enriched process in LSECs being blood vessel
morphogenesis ([Fig. 2A ]). IPA disease and function showed enrichment in LSECs in categories of cell death
and survival, cardiovascular system development and function, and inflammatory response.
Therefore, this further emphasizes the clear biofunctional differences that distinguish
LSECs from hepatocytes.
Fig. 2 Comparison of LSECs and hepatocytes. (A ) Significant enriched ontologies of the genes that are showing difference in expression
for biological process. (B ) Disease and functions identified for differentially expressed genes between LSEC
fetal and other fetal hepatocytes. (Detail of the figure as described in [Fig. 1 ]). LSECs, liver sinusoidal endothelial cells.
Expression of Coagulation Factors in Different Cell Types
Expression of Coagulation Factors in Different Cell Types
Each one of the protein/factors involved in coagulations is synthesized in specific
cell type. The hepatocytes constitute the main site of synthesis for most of the coagulation
factors that include fibrinogen, FII, FV, FVII, FIX, FX, FXI, FXII, and ProtC. Other
coagulation proteins are made by hepatocytes and some endothelial cells such as TFPI
and PROS1. Most endothelial cells including LSECs synthesize FII receptor and VWF.
Factor VIII protein is clearly synthesized in LSECs and neither in other endothelial
cells and nor in hepatocytes ([Fig. 3 ]).
Fig. 3 Expression of different coagulation factors in fetal endothelial cells, fetal liver
sinusoidal endothelial cells, and hepatocytes. From left to right: heat map—representing
expression in each sample, sample principle component analysis (PCA) plot, and variable
PCA plot.
Conclusion
Hemophilia A continues to be an exceptional genetic disease with excellent treatment
options. In the past 5 years, recombinant FVIII protein factor concentrates with improved
pharmacokinetics and most recently a FVIII mimetic bispecific antibody became available
for the efficient treatment of hemophilia patients. However, these options can only
raise through levels to 5 to 15 IU/dL and still depend on regular subcutaneous and/or
intravenous injections. Gene therapy when effective will allow hemophilia A patients
to have an almost normal life. Although current gene therapy protocols have shown
to correct the bleeding phenotype, especially in hemophilia A, long-term immune suppression,
variability, duration, and long-term safety remain important limitations. Therefore,
optimizing gene therapy protocols remains of special significance. Because of specific
characteristics of cell types, targeting the natural cell of FVIII synthesis may represent
one of the future strategies in hemophilia A.
