Cell mediated gene therapy: A guide for doctors in the clinic

Table 1 Current cell mediated gene therapy protocols in clinical and R and D stages

Target disease	Stage	Gene modification	Target cell	Ref.
Hemophilia A	Preclinical	Oct4, Sox2, Klf4	Fibroblast	Xu et al[40]
ALS	Preclinical	Oct4, Sox2, Klf4, c-Myc	Fibroblast	Dimos et al[55]
Alzheimer	Preclinical	Oct4, Sox2, Klf4, c-Myc	Fibroblast	Israel et al[46]
ALS	Preclinical	Oct4, Sox2, Klf4	Fibroblast	Chen et al[54]
Cardiac failure	Preclinical	Gata4, Mef2c, Tbx4	Fibroblast	Ieda et al[22]
Diabetes	Preclinical	Oct4, Sox2, Klf4	Fibroblast	Zhang et al[24]
Schizophrenia	Preclinical	Oct4, Sox2, Klf4, c-Myc	Fibroblast with 15q11.2 del	Yoon et al[12]
Barth syndrome	Preclinical	Oct4, Sox2, Klf4, c-Myc	Fibroblast	Wang et al[52]
Parkinson’s disease	Preclinical	Oct4, Sox2, Klf4, c-Myc	Fibroblast	Chung et al[50]
Macular degeneration	PhaseI	Oct4, Sox2, Klf4, c-Myc	Fibroblast	Kamao et al[14]
Degenerative arthritis	Phase II/III	TGF-β1	Chondrocyte	Ha et al[15]
X-linked adrenoleukodystrophy	In the clinic	ABCD1	CD34+ Bone marrow cell	Cartier et al[36]
Wiskott-aldrich syndrome	In the clinic	WASP	CD34+ Bone marrow cell	Aiuti et al[35]
Metachromatic leukodystrophy	In the clinic	ARSA	CD34+ Bone marrow cell	Biffi et al[37]

ALS: Amyotrophic lateral sclerosis; TGF-β: Transforming growth factor-beta.

Table 2 Different types of production methods based on cells needed per batch

Cells/lot (billions)	Cell culture	Harvest	Filling	Freezing
1	T175 flask or hyperflask, 10 layer cell factory	Bucket centrifugation	Hand/manual fill	Bench top control rate freezer
10	10 layer cell factory, hyperstack-12, Xpansion systems	Bucket centrifugation	Semi automated fill machine or hand/manual fill	Bench top control rate freezer
50	Hyperstack or Xpansion, cell cube	Tangential flow filtration, continuous centrifugation	Automated fill machine	Large scale control rate freezer
100	Factory automation of Xpansion or Hyperstack technologies	Tangential flow filtration of continuous centrifugation	Automated fill machine	Large scale control rate freezer
500	Bioreactors using microcarriers	Continuous centrifugation	Automated fill machine	Scale out large scale control rate freezer

Table 3 Suggested release tests advised by Food and Drug Administration for cell product

Category	Test	Key measurement
Sterility	Sterility	Microbiological testing
	Mycoplasma	After pooling of cultures but before washing
Identity	Identity	Cell identity and assess heterogeneity
Purity	Impurity	Residuals contaminants
	Endotoxin	Contaminants during the process
Potency	Specific to product	Relevant function of the cells
Other testing	General safety	Cellular therapy products are exempt
	Viability	Generally > 70%
	Cell number	Minimum and maximum cell number
Title 21 of the code of federal regulations applied to cell therapy products
The tissue rules: Part 1271
The biologics requirements: Part 600 and 610
The investigational new drug requirements: Part 312
The drug manufacturing requirements: Parts 211 and 212

Table 4 TG-C release test

Test	Method	Specification
Release tests and specifications for hChonJ cell
Identification
Cell growth and morphology	Visual (test code 30117)	Fibroblast-like: spindle shaped/bipolar or multipolar cells
Viability	Trypan blue dye exclusion	> 70%
	(test code 30458)
Mycoplasma	1993 Points to consider (test code 30055)	Negative
RAP test	In vivo and ELISA (test code 30163)	Negative
Sterility (direct inoculation method)	21 CFR 610.12 (test code 30744)	Negative
Endotoxin	LAL (test code 37653)	< 5 EU/mL
Release tests and specifications for hChonJb#7 Cell
Identity		Epithelial-like: fried egg or polygonal-shaped appearance
Cell growth and morphology	Visual (test code 30117)
Type II collagen assay	RT-PCR (test code 30412)	Type II collagen present
TGF-β1 presence	PCR (test code 30959)	TGF-β1 present
Potency (TGF-β1 assay)	ELISA (test code 30444)	1-50 ng/105 cells/24 h
Viability	Trypan blue dye exclusion (test code 30458)	> 70%
RCR	Co-culture of end of production cells (test codes 30628)	Negative
	Supernatant amplification (test code 30633)	Negative
Mycoplasma	1993 points to consider (test code 30055)	Negative
Rat antibody production	In vivo and ELISA (test code 30163)	Negative
Endotoxin	LAL (test code 37653)	< 5 EU/mL
Sterility	21 CFR 610.12 (test code 30744)	Negative

RCR: Replication competent retrovirus; LAL: Limulous amoebocyte lysate; RT-PCR: Reverse transcription polymerase chain reaction; CFR: Code of federal regulation; RAP: Rat antibody production.