SAFETY CONCERNS

SAFETY CONCERNS

β 3 was The major safety concerns of cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of the most serious concerns is that of tumour formation and malignant transfor - mation. The risk of tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of stem cells to form tera tomas is one of the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF

TABLE 4.3 Risks of cell-based therapy. Tumour formation Genetic and epigenetic abnormalities Transmission of infection Poor viability and loss of function Differentiation to undesired cell types Rejection (allogeneic cells) Side effects of immunosuppression (allogeneic cells)

α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of pluripotency , and the risk of this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of viral vectors that do not integrate into the genome or of non-viral approaches to di ff erentiation reduces the risk of malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of transmitting infection. It is essential that if allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .

Group 1 Group 2 Group 3 Intact Infection G/P (pretreatment) Day 16 Day 16 Day 16 Day 2 SMA SMA SMA SMA DAPI DAPI DAPI DAPI (b) DAPI DAPI DAPI DAPI (c) DAPI DAPI DAPI DAPI Figure 4.10 Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel /f_l uid gel carrier (FG) (group 2) and standard of care plus the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring: (c) laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi /f_i cant reduction in scar markers. DAPI, 4 ,6-diamidino-2-phenylindole ( /f_l uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted with permission from Hill LJ, Moakes RJA, Vareechon C et al . Sustained release of decorin to the surface of the eye enables scarless corneal regeneration. npj Regen Med 2018; 3: 23.) G/P/FG G/P/DecFG 120 100 80 60 40 20 0 SMA DAPI 120 100 80 60 40 20 0 DAPI 120 100 80 60 40 20 0 DAPI (a) -smooth muscle actin ( -SMA), (b) /f_i bronectin and

Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of e ffi cacy , by way of well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of patient subgroups and staging of disease. In addi tion to clinical studies and evidence , the rapid pace of therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing. SAFETY CONCERNS

β 3 was The major safety concerns of cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of the most serious concerns is that of tumour formation and malignant transfor - mation. The risk of tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of stem cells to form tera tomas is one of the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF

TABLE 4.3 Risks of cell-based therapy. Tumour formation Genetic and epigenetic abnormalities Transmission of infection Poor viability and loss of function Differentiation to undesired cell types Rejection (allogeneic cells) Side effects of immunosuppression (allogeneic cells)

α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of pluripotency , and the risk of this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of viral vectors that do not integrate into the genome or of non-viral approaches to di ff erentiation reduces the risk of malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of transmitting infection. It is essential that if allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .

Group 1 Group 2 Group 3 Intact Infection G/P (pretreatment) Day 16 Day 16 Day 16 Day 2 SMA SMA SMA SMA DAPI DAPI DAPI DAPI (b) DAPI DAPI DAPI DAPI (c) DAPI DAPI DAPI DAPI Figure 4.10 Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel /f_l uid gel carrier (FG) (group 2) and standard of care plus the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring: (c) laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi /f_i cant reduction in scar markers. DAPI, 4 ,6-diamidino-2-phenylindole ( /f_l uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted with permission from Hill LJ, Moakes RJA, Vareechon C et al . Sustained release of decorin to the surface of the eye enables scarless corneal regeneration. npj Regen Med 2018; 3: 23.) G/P/FG G/P/DecFG 120 100 80 60 40 20 0 SMA DAPI 120 100 80 60 40 20 0 DAPI 120 100 80 60 40 20 0 DAPI (a) -smooth muscle actin ( -SMA), (b) /f_i bronectin and

Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of e ffi cacy , by way of well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of patient subgroups and staging of disease. In addi tion to clinical studies and evidence , the rapid pace of therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing. SAFETY CONCERNS

β 3 was The major safety concerns of cell-based therapy and tissue β 3 engineering are listed in Table 4.3 . One of the most serious concerns is that of tumour formation and malignant transfor - mation. The risk of tumour formation varies according to the - cell type used, the genetic modification strategy used to trans - form the stem cells, the site of transplantation and w hether the cells are autologous or allogeneic. The direct risk of tumour formation by the transplanted cells relates specifically to ESCs and iPSCs and there appears to be little risk with SSCs. The ability of stem cells to form tera tomas is one of the hallmarks /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF /uni25CF

TABLE 4.3 Risks of cell-based therapy. Tumour formation Genetic and epigenetic abnormalities Transmission of infection Poor viability and loss of function Differentiation to undesired cell types Rejection (allogeneic cells) Side effects of immunosuppression (allogeneic cells)

α - α - α - α - Fibronectin Fibronectin Fibronectin Fibronectin Fibronectin Laminin Laminin Laminin Laminin Laminin /uni2032 of pluripotency , and the risk of this happening following stem cell therapy may be reduced by ensuring that only cells that have been fully di ff erentiated in vitro , and not those that are still pluripotent, are used for therapy . The risk of malignancy may also be reduced by the choice of in vitro strategy used to di ff erentiate stem cells prior to use: the use of viral vectors that do not integrate into the genome or of non-viral approaches to di ff erentiation reduces the risk of malignant transformation. There is also interest in developing techniques for directly reprogramming somatic cells to adopt the function of a the threshold (%) Percentage of pixels above t tac n I α - tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr P = 0.051 the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr - the threshold (%) Percentage of pixels above t tac n I tion (day 2) ec nf oup 1 (day 16) oup 2 (day 16) oup 3 (day 16) I Gr Gr Gr α α di ff erent cell type without having to make them first revert back to the pluripotent state – so-called transdi ff erentiation. Another major concern is that of transmitting infection. It is essential that if allogeneic stem cells are used they are screened to exclude infection and that cells and engineered tissues ar e prepared accor ding to GMP guidelines to avoid bac - terial infection during in vitro culture prior to use. Moreover, if allogeneic cells are used for tissue engineering and regenerative therapy , they may be susceptible to graft rejection and immuno - suppressive therapy may be necessary .

Group 1 Group 2 Group 3 Intact Infection G/P (pretreatment) Day 16 Day 16 Day 16 Day 2 SMA SMA SMA SMA DAPI DAPI DAPI DAPI (b) DAPI DAPI DAPI DAPI (c) DAPI DAPI DAPI DAPI Figure 4.10 Stained sections through a mouse cornea before (left) and after (second column) injury and subsequent treatment with standard of care (gentamicin [G] and prednisolone [P]; group 1), standard of care plus a novel /f_l uid gel carrier (FG) (group 2) and standard of care plus the carrier and decorin (Dec) (group 3). Sections are stained for markers of scarring: (c) laminin. Importantly, the use of the slow-release decorin resulted in rapid restoration of the corneal structure with a signi /f_i cant reduction in scar markers. DAPI, 4 ,6-diamidino-2-phenylindole ( /f_l uorescent stain that binds strongly to adenine-/thymine-rich regions in DNA). (Adapted with permission from Hill LJ, Moakes RJA, Vareechon C et al . Sustained release of decorin to the surface of the eye enables scarless corneal regeneration. npj Regen Med 2018; 3: 23.) G/P/FG G/P/DecFG 120 100 80 60 40 20 0 SMA DAPI 120 100 80 60 40 20 0 DAPI 120 100 80 60 40 20 0 DAPI (a) -smooth muscle actin ( -SMA), (b) /f_i bronectin and

Tissue engineering and regenerative strategies hold out great hope for e ff ectively repairing or replacing tissues in a wide number of human diseases. The field is moving rapidly , under pinned by new developments in the relevant science in stem cells, materials and molecules. New emerging areas of tech nology include therapeutic signalling by wa y of extracellular vesicles (EVs) and gene editing of cells using CRISPR-Cas9, and gene therapies. All will require the use of a translational approach, wher eby the hypothesised mechanism is developed and translated to the clinic, building up robust clinical evidence of e ffi cacy , by way of well-designed and well-conducted clinical trials before widespread adoption. It is likely that patient stratification will further refine ther apy options. The ability to phenotype, genotype and profile patients at a molecular level will allow more detailed charac terisation of patient subgroups and staging of disease. In addi tion to clinical studies and evidence , the rapid pace of therapy development will need to be accompanied by the development of new regulatory frameworks, f or example in point-of-care manufacturing.