Flashcards in Stem cell therapy for the treatment of osteogenesis imperfecta Deck (32):
what is OI?
brittle bone disease- fragile bones at break very easily and bend. babies normally die.
how common is OI?
1 in 10,000
what gene is mutated in this disease?
type I collagen
how many different mutations are there that give rise to OI?
what does the mutation in collagen result in?
abnormal bone ECM- collagen gives bones their strength and having the wrong type can result in flexible bones
is OI visible before birth?
what is the rationale behind using stem cells to treat OI?
- stem cells are undifferentiated, stem cells self-renew and are expandable in vitro, MSCs are precursors to bone forming cells- osteoblasts
why are MSCs good cells types to use for clinical uses?
- they differentiate readily into osteoblasts
when would stem cells be introduced? what are the 4 reasons for this
- intraterine injection. The stem cells would deb given before its immune competence has reached maturity. give cells before irreversible damage. The metal environment i very permissive to prenatal stem cell transplantation. Also if you have a smaller baby then you need lower cell numbers to expand
in the clinical trial that is being undertaken, where are the MSCs derived from?
the fetal liver- put into the OI foetus.
why are MSCs used?
they can differentiate into osteoblasts
where do the stem cells come from dying these clinical trials?
they come from a healthy donor that has no genetic condition - from a foetus because foetal stem cells are different to human cells- y have loner telomeres, can proliferate faster, better at migrating and better at differentiating.
what mouse model has been used to look at OI?
they use a mouse which has a mutation that disrupts the protein and therefore the collagen 1 alpha 2 is not formed. and so collagen becomes ahomodimer rather than heterodimer ( of collegen 1alpha 1. This results in a brittle bone phenotype
what is the experimental procedure for using mice?
- they used human MSCs in a mouse model
- it is injected into the pregnant mouse by taking the uterine horn out of the mummy mouse and then injected into each other the babies.
- then at 21 days the babies are born.
how did they measure the success of transplanting man MSCs into foetal mice?
- they looked at the fracture rate in mice at different ages. there is a massive reduction.
- the tested the mechanica strength: looked at the 3 point bending test. You find that the bones are less brittle.
- they looked at the cortex of the bone and measured the thickness of the cortex and this is thicker- has an improved structure too then.
- the donor cells express collagen 1 alpha 2- they looked to see if it was present in these mutants you find they are present- they survived, they migrated to the bone and then became osteoblasts ( the specific markers such was osteocalcin)
what is found about the engraftment of the MSC donor cells?
they preferentially migrate to the site of injury at the bone fracture sites where they accumulate in the bones and differentiate which is very good
what is the general premise of using stem cells to treat OI?
the stem cells replace the mutated genes- so the donors can produce normal collagen fibres
what are the areas of further study?
- have to get these cells from aborted embryos so not good. would like to have access to stem cells during the pregnancy and alternative cells tat have the same properties- placenta cells.
what is an interesting approach to sing gene therapy to treat it and why?
Although the resolutive cure for dominant OI would be the correction of the DNA defect, for the more severe forms the silencing of the mutant allele may represent an interesting option. The specific suppression of the mutant allele, with the consequent haploinsufficiency, will con- vert a severe phenotype characterized by collagen struc- tural defects to a mild OI, caused by a quantitative defect.
what techqniues can been used for gene therapy to silence the mutant allele and hence turn a qualitative trait into haploinsufficiency which is milder?
everal attempts in this direction had been made using various antisense technologies such as antisense oligo- deoxyribonucleotides (ODNs), ribozymes, short interfering RNA (siRNA), and short hairpin RNA (shRNA) (Fig. 1ac) (25). Although efficiency and specificity were not absolute, promising data were obtained for all the silencing tools tested. The majority of the published reports comes from studies using either in vitro or ex vivo pproaches, and less data are available from in vivo studies in OI murine models (for dominant OI)
give a precise example of an experiment seeking to silence the col1a1 allele
In these cells, shRNAs subcloned in a lentiviral vector and stably integrated into the endogen- ous genome reduced the amount of mutant transcript by 5765% versus 2428% of wild-type transcripts.
how could ipscs be used for this disease treatment?
- could correct the gene by isolating MScs fro OI patients and generation of iPSCs derived from patients’ MSCs following in vitro correction were able to differentiate to osteoblast and to produce normal collagen and successfully formed bone in vivo (46). The silencing of the mutant allele in induced patient’s iPSCs able to differentiate to osteoblasts with subsequent return of the modified cells to the affected individual will represent an exciting avenue in OI treatment.
(for less extreme cases)
what is the premise of using cell therapy?
The scientific base of the treatment is the absence of clinical skeletal phenotype in mosaic carriers even in presence of a high number of mutant osteoblasts (47). This observation suggested that even a small fraction of normal osteoprogenitor cells may lead to significant phenotypic benefit
what is the major drawback of using cel ltherapy to treat not fatal cases?
The challenge for OI gene and stem cells therapy will be to specifically target the bone tissue. To date a high therapeutic dose of systemically administered modifying molecule or a high number of transplanted cells would be needed to stimulate sufficient bone formation and may carry a high risk for adverse effects in non-skeletal tissues
- For systemic delivery of therapeutic nucleic acids, viral vectors have been used because of their high transfection efficacy, but the severe safety risks due to their oncogenic potential and their inflammatory and immunogenic effects prevent them from repeated administration (66, 67). siRNA delivery systems made in the last years a rapid progress, but bone remains a difficult target tissue to be reached
what is the current treatment of care for OI?
- biphosphonates to improve bone strength by inhibiting bone resorption but they dont reduce fracture indicidence long term
describe 2 therapies in humans that have been successful
- whole bone marrow and bone marrow mesenchymal stem cells ave been transplanted in OI children with gains in body length and bone mineralisation
- allogenic liver-derived stem cells transplanted in utter led to apparent phonetic imporvment in an OI foetus
what has been a really rpimising source of stem cells for the treatment of this disease? how has this been tested?
human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification.
what are the problems with using fetal liver MSCs?
- there are ethical concerns and are not infinite in supply
what is the main genetic cause of oI?
Point mutations in either COL1A1 or COL1A2 resulting in substitutions of a glycine residue or in an aberrant splicing are mainly responsible for the altered type I collagen structure found in the moderately severe OI type IV, progressive deforming OI type III and lethal OI type II
how can whole bone marrow be used to treat OI?
mesnehcymal stem cells are found in the bound marrow so you can actually do a bone marrow transplant
why are mesenchymal stem cells used?
they are able to differentitate into osteoblasts (bone cells),chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes