FUTURE PROSPECTS FOR CELL & MOLECULAR THERAPY

what is cell-based therapy

the administration of cells as ‘living agents’ in patients to fight disease

what are challenges of cell therapy

• identification of the appropriate cell source

• generation of a sufficiently viable, potent and safe product

• development of scalable manufacturing processes

from what sources can cells be derived from in cell based therapy

• autologous cells

• allogeneic cells

• xenogeneic cells

outline autologous cells

• patient’s own cells

  • bone marrow-derived haematopoietic stem cells (HSCs)

  • immune effector cells isolated from peripheral blood

  • induced pluripotent stem cells (iPSCs)

what are advantages and disadvantages of autologous cells

advantages

• avoid immune response (good long-term engraftment)

• iPSCs can turn cells that have terminally differentiated into pluripotent cells

disadvantages

• dependence of product quality upon the patient’s health

• high manufacturing costs

outline allogeneic cells

• human origin but from an individual distinct from the patient

• cells usually encapsulated in biopolymer matrices to prevent immune recognition

what are advantages and disadvantages of allogeneic cells

advantages

• potentially scalable production

• source will be a healthy human being so cells will be of high quality

• theoretically unlimited supply

what are examples of allogeneic cells

• natural killer cells (NK)

• mesenchymal stem cells (MSCs)

outline xenogeneic cells

• of non-human origin

• often porcine cells

what are challenges of using xenogeneic cells in cell-based therapy

• overcoming host immune rejection

• patients need immunosuppressants for the rest of their life

  • often they are very sick already so giving them immunosuppressants weakens their immune system even further so they are more likely to get sick again

• some patients may not be able to receive porcine cells due to religious/ lifestyle

image showing challenges at each stage of the cell therapy production process

table showing dental application of cell-based therapies

outline Gintuit

• made of isolated living cells from human skin cells (keratinocytes) and grown with bovine collagen

• cross-talk between cell release growth factors and cytokines that promote tissue regeneration

• replace palatal grafts used for receding gums

• available in US not UK

what advantage does Gintuit have over the traditional gold standard graft method

• Gintuit is much better tolerated by the patient

• traditionally, tissue was grafted from the palate but this is very painful

outline CAR T-cell therapy

• precision medicine: type of cancer immunotherapy

• T-cells are re-engineered to produce chimeric antigen receptors (CAR) that recognise antigens of malignant cells

• ‘giving patients a living drug’

what are the risks of CAR T-cell therapy

• cytokine release syndrome (CRS) due to large immune response because T-cells release cytokines

  • patients feel severe fatigue, fever and breathing difficulties

for CAR T-cell therapy to work, it must be known what cancer the patient has, why is this?

• patient’s own T-cells are isolated

• T-cells are modified to recognise the specific antigens produced by tumor cells

• therefore must know what cancer the patient has

list of FDA approved CAR T-cell therapies

give an example of autologous based therapies

• stem cell derived insulin-producing cells can theoretically be generated in endless quantities » large amount of insulin generated

• ongoing clinical trials of stem cell based therapies to cure type 1 diabetes

• transplants pluripotent stem cell derived pancreatic endoderm cells in macroencapsulation devices which can be vascularised to receive signals from the human body

—

• CT do not prove therapeutic levels of insulin secretion or provide unequivocal evidence of clinical benefit

• therapeutic levels of insulin produced was not enough when tested in human patients

what can cell therapy be theoretically used for in dentistry

• use of dental stem cells for tooth regeneration

• biomaterials/ scaffolds for regenerative dentistry (maxillofacial incl. soft tissues)

• bioengineered tooth erupted and physiologically similar to natural teeth

• autologous transplantation of bioengineered tooth germ reconstructed using a patient’s own stem cells

what cell types can be derived from dental stem cells

a) cementoblast

b) adipocyte

c) odontoblast

d) neuronal cells

e) myoblast

f) chondrocyte

g) pulp cells

h) hepatocyte

i) endothelial cell

j) osteoblast

k) melanocyte

give an example of a clinical application of stem cell therapy

• use of deciduous dental pulp stem cell (DDPSC) for unilateral cleft defects

• outcomes: significant results of bone regeneration compared with traditional methods of bone grafting

Tanikawa DYS et al. (2020)

what other condition can stem cell therapy be used to treat

apexification (procedure that closes the tip of an open apex of an immature tooth with a non-vital pulp)

what are the main challenges of cell therapy in dentistry

• achieving the correct type, size, shape and colour of the original tooth

• achieving full function of the regenerated tooth with vascularisation, innervation and binding to supporting tissues

what is tolerance induction

tolerance induction: giving the patient a little at a time

how can cell therapies be modified

• using biomaterials

• using gene editing

outline biomaterials in cell therapy modifications

• improves the delivery, viability, retention and safety of therapeutic cells

• encapsulation is effective in preventing immediate rejection of allogeneic cells

• long-term effect is a challenge due to foreign body response

how can graft cell survival be improved

• changes in physical properties

  • size

  • shape

  • surface

  • morphology

  • roughness

  • topography

  • geometry

outline gene editing in cell therapy modifications

• cell-based therapies will almost certainly progress with the aid of genome and epigenome editing tools i.e. gene therapy

• engineering approaches:

  • genetic and/ or epigenetic modification

  • CRISPR and CRISPR-associated (Cas) proteins

    • double strand breaks (DSBs)

what are the concerns for engineered cells in gene editing (cell therapy modifications)

• DSBs can cause harmful genomic rearrangements

• CRISPR-Cas-based based editors (without creating a DSB)

what is gRNA

gRNA = guide RNA

• with Cas 9 so Cas 9 knows where to cut the genome

what are problems with CRISPR-Cas9

• CRISPR-Cas9 gene editing in early human embryos leads to frequent loss of the targeted chromosome

• this indicated that human germline gene editing would pose a substantial risk for aneuploidy (abnormal number of chromosomes in a cell)

• therefore cannot be used in vivo safely

what is a major issue with CRISPR technology

• deliberately producing double stranded breaks means repairs can go wrong

• there are also lots of repetitive areas of DNA so if gRNA is produced incorrectly, there can be a lot of double stranded breaks in DNA

what is gene therapy

a technique for correcting defective genes responsible for disease development e.g. replace a missing/ mutated gene, add genes to fight the disease, turn off genes causing diseases

• vectors deliver genes to patient’s target cells

  • vectors are commonly viruses (retro, adeno or adeno associated)

• introduction of a foreign piece of DNA into genome

what does gene therapy success depend on

• good molecular strategy and a safe, efficient and specific gene delivery system

  • viral vectors - highest efficiencies but are associated with immunogenicity

  • non-viral vectors - safer but not as efficient

outline ex vivo gene therapy

• tissue cells from patient are cultured and transduced with modified vector (cells are treated with virus)

• transduced cells that produce therapeutic protein are infused back into the body’s genome (transfection)

outline in vivo gene therapy

• the modified vector is directly injected into the body

• the therapeutic gene is inserted into the virus, virus is injected directly into the body

outline gendicine

• first commercially available gene therapy in 2003 approved by China FDA

• recombinant human p53 adenovirus - injected directly into tumour and induces tumour cell apoptosis, senescence or autophagy

• used mainly for head and neck squamous cell carcinoma (intra-tumoral injection)

recent example of in vivo gene therapy

adds back aquaporin » improved saliva flow rate and tear flow

what are challenges in gene therapy

• toxicity, immune and inflammatory responses of viral vectors

• DNA integration into the genome

• many human diseases are multigene disorders so gene therapies that target multiple genes need to be developed

  • seem effective in monogeneic conditions

• ethical aspect

outline the ethical challenges in gene therapy

• how can ‘good’ and ‘bad’ uses of these technologies be distinguished?

• who decides which traits are normal and which constitute a disability or disorder?

• will the high costs of gene therapy make it available only to the wealthy?

• could the widespread use of gene therapy make society less accepting of people who are different?

• should people be allowed to use gene therapy to enhance basic human traits such as height, intelligence or athletic ability?

state the differences between somatic VS germline gene editing

other molecular therapies