Stem cell therapy for the treatment of osteogenesis imperfecta

Question 1

what is OI?

Answer 1

brittle bone disease- fragile bones at break very easily and bend. babies normally die.

Question 2

how common is OI?

Answer 2

1 in 10,000

Question 3

what gene is mutated in this disease?

Answer 3

type I collagen

Question 4

how many different mutations are there that give rise to OI?

Answer 4

250

Question 5

what does the mutation in collagen result in?

Answer 5

abnormal bone ECM- collagen gives bones their strength and having the wrong type can result in flexible bones

Question 6

is OI visible before birth?

Answer 6

yes

Question 7

what is the rationale behind using stem cells to treat OI?

Answer 7

• stem cells are undifferentiated, stem cells self-renew and are expandable in vitro, MSCs are precursors to bone forming cells- osteoblasts

Question 8

why are MSCs good cells types to use for clinical uses?

Answer 8

• they differentiate readily into osteoblasts

Question 9

when would stem cells be introduced? what are the 4 reasons for this

Answer 9

• 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

Question 10

in the clinical trial that is being undertaken, where are the MSCs derived from?

Answer 10

the fetal liver- put into the OI foetus.

Question 11

why are MSCs used?

Answer 11

they can differentiate into osteoblasts

Question 12

where do the stem cells come from dying these clinical trials?

Answer 12

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.

Question 13

what mouse model has been used to look at OI?

Answer 13

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

Question 14

what is the experimental procedure for using mice?

Answer 14

• 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.

Question 15

how did they measure the success of transplanting man MSCs into foetal mice?

Answer 15

• 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)

Question 16

what is found about the engraftment of the MSC donor cells?

Answer 16

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

Question 17

what is the general premise of using stem cells to treat OI?

Answer 17

the stem cells replace the mutated genes- so the donors can produce normal collagen fibres

Question 18

what are the areas of further study?

Answer 18

• 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.

Question 19

what is an interesting approach to sing gene therapy to treat it and why?

Answer 19

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.

Question 20

what techqniues can been used for gene therapy to silence the mutant allele and hence turn a qualitative trait into haploinsufficiency which is milder?

Answer 20

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. 1a􏲒c) (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)

Question 21

give a precise example of an experiment seeking to silence the col1a1 allele

Answer 21

In these cells, shRNAs subcloned in a lentiviral vector and stably integrated into the endogen- ous genome reduced the amount of mutant transcript by 57􏲒65% versus 24􏲒28% of wild-type transcripts.

Question 22

how could ipscs be used for this disease treatment?

Answer 22

• 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)

Question 23

what is the premise of using cell therapy?

Answer 23

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

Question 24

what is the major drawback of using cel ltherapy to treat not fatal cases?

Answer 24

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

Question 25

what is the current treatment of care for OI?

Answer 25

• biphosphonates to improve bone strength by inhibiting bone resorption but they dont reduce fracture indicidence long term

Question 26

describe 2 therapies in humans that have been successful

Answer 26

• 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

Question 27

what has been a really rpimising source of stem cells for the treatment of this disease? how has this been tested?

Answer 27

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.

Question 28

what are the problems with using fetal liver MSCs?

Answer 28

• there are ethical concerns and are not infinite in supply

Question 29

what is the main genetic cause of oI?

Answer 29

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

Question 30

how can whole bone marrow be used to treat OI?

Answer 30

mesnehcymal stem cells are found in the bound marrow so you can actually do a bone marrow transplant

Question 31

why are mesenchymal stem cells used?

Answer 31

they are able to differentitate into osteoblasts (bone cells),chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes

Question 32

what is the link between osteoblasts, collagen mutation and the phenotype seen in the disease?

Answer 32

Analyses of the cellular contribution to bone properties from two mice models of OI suggested that collagen mutations exert effects on the bone cellular function and survival [