11 - Whole Cell Gene Therapy

Question 1

Duchenne Muscular Dystrophy (DMD)

Answer 1

• Result of non functional dystrophin gene
• X linked recessive disease
• Wheelchair bound by 12 with death due to cardiac or respiratory failure
• mdx mouse is a model of this disease
• nonsense, frameshift and in frame mutations

Question 2

Dystrophin

Answer 2

• Gene is 2.4 Mb long
• 79 exons
• Encodes multiple isoforms

Question 3

Genetic therapy approaches to DMD

Answer 3

• Mini dystrophin gene delivery
• Transcriptional read through
• Exon skipping
• Homologue induction
• Cell based whole gene replacement

Question 4

Mini dystrophin gene delivery

Answer 4

Smaller dystrophin genes based on Becker examples can be delivered via plasmid and viral vectors

Question 5

Transcriptional read through

Answer 5

Some antibiotics will cause ribosomes to ignore stop codons

Question 6

Exon tripping

Answer 6

Intron splicing mechanism can be used to create a becker-like messenger mRNA in vivo

Question 7

Homologue Induction

Answer 7

A dystrophin analogue called utrophin has a promoter which
may allow selective upregulation

Question 8

Cell based whole gene replacement

Answer 8

Donor myoblasts which have functional dystrophin gene can be transplanted by Myoblast Transfer Therapy

Question 9

Skeletal Muscle Formation

Answer 9

Myoblasts > Myotubes > Myofibre

Question 10

Myoblast Transfer Therapy (MTT)

Answer 10

Direct injection of dystrophin expressing donor myoblasts into dystrophic muscle in order to provide a cell based gene rescue

Question 11

The Immune Response and Myoblast Survival

Answer 11

• Little role of innate immune system and humoral immunity
• MTT in immunosuppressed, nude and SCID mice exhibited superior myoblast survival.
• The acquired immune system implicated in poor donor myoblast survival

Question 12

Tregs

Answer 12

CD4+CD25+ T cells essential for the
control of autoreactive T cells in vivo

Question 13

Tregs mechanism of action

Answer 13

• Suppress immune responses via cell to cell interactions and cytokine production
• IL-10 and TGF-b implicated in Treg induction and suppressive function

Question 14

Mammalian Expression Vector pMP6a

Answer 14

• Bacterial origin of replication (can be expanded in bacteria)
• Ampicillin resistance gene (select for bacteria that have been transformed)
• Cytomegalovirus promotor (can be expressed in eukaryotic cells)
• Unique restriction site after promoter (where genes of interest are added)

Question 15

Transformation of E.coli by Electroporation

Answer 15

• Mix bacteria and plasmid
• In vitro electroporation
• Transfection

Question 16

Plasmid amplification and purification

Answer 16

• Transformation mixture added to media for 1hr non selective regrowth
• Spread plate regrowth onto ampicillin agar plates
• Colonies result from transformed cells containing plasmid
• Pick single colony and grow in overnight culture
• Conduct plasmid isolation

Question 17

Purity of Plasmid Preparations

Answer 17

RE digestion with Nhe1

Question 18

Cytokine Expression in Mammalian Cells

Answer 18

• Transfect eukaryotic cells with pMP6a plasmids
• Tissue Culture cells for 48 hrs
• Quantitate cytokine expression by ELISA

Question 19

In vivo Electroporation of Muscle

Answer 19

• Inject plasmid
• In vivo electroporation
• Transfection and cytokine expression

Question 20

Disappointing Outcomes in Muscle Targeted Cytokine Therapy and Tregs

Answer 20

• The recombinant shuttle plasmid gene therapy induced Tregs in dystrophic muscle and should have, in principle, led to less rejection following MTT
• Repeated experiments showed no improvement in transplanted myoblast survival
• The answer as to why this approach has failed may lie with microenvironmental factors