THE PAST, PRESENT,

AND FUTURE OF
CELL AND GENE
THERAPY

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Starting Blocks: An Gathering Momentum: Top Speed: the Future of Gene Manipulation 101
Introduction to Cell and the Current State of Cell Cell and Gene Therapy
Gene Therapy and Gene Therapy
An End-to-End Cell & Gene
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1. Center for Biologics Evaluation and Research. Approved cellular and gene therapy products. Retrieved July 01, 2021, from
https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapyproducts/approved-cellular-and-gene-therapy-products
 STARTING BLOCKS:
AN INTRODUCTION
TO CELL AND GENE
THERAPY
S
ince Frederick Griffith’s dis-
covery of the transforming
principle in 1928, scientists
have recognized that cells
can change their properties
by taking up exogenous genetic mate-
rial.1 This principle shaped the founda-
tion of gene therapy: manipulating genes
could be used to treat diseases. While
gene therapy started with a relatively
narrow scope—the treatment of hered-
itary illnesses or diseases with underly-
ing genetic causes—it now has a much
broader application potential.
Progress and Tragedy
Griffith’s discovery was followed by the
characterization of DNA and the discov-
ery that viruses could be used to deliver
genetic material to cells. This knowl-
edge paved the way for the first example
of heritable gene transfer in mammalian
cells in 1962.1 From there, the seventies
saw the first human gene therapy clin-
ical trials, which aimed—unsuccess-
fully—to introduce the gene encoding
arginase into two girls suffering from a
urea cycle disorder.1
It took until 1990 for the first FDA-ap-
proved gene therapy clinical trial to take
place. Using recombinant DNA, white
blood cells from patients suffering from
adenosine deaminase deficiency (ADA-
SCID) were modified ex vivo to enable
them to produce adenosine deaminase.
While patient response was mixed, this
first step spurred similar trials in the
European Union for ADA-SCID and sev-
THE PAST, PRESENT, AND FUTURE OF CELL AND GENE THERAPY
eral other diseases.1 Unfortunately, the
gene therapy boom was quickly halted
in 1999 by the death of Jesse Gelsinger,
an 18-year-old who received adenoviral
gene therapy to treat a partial deficiency
of ornithine transcarbamylase (OTC).1
Understanding and Mitigating
the Risk
Gelsinger died from multi-organ fail-
ure caused by an immune response to
the adenovirus vector used during treat-
ment, highlighting the risks potentially
posed by gene therapy viral vectors.1
This led researchers to consider trans-
ducing a patient’s cells ex vivo and then
re-introducing them to the host’s body,
which would prevent deleterious immune
responses and remove the need to inject
viruses into patients. As such, gene ther-
apy and cell therapy quickly formed a
symbiotic relationship.
Cell therapy dates back to the 19 th
century, but for the majority of its
history, scientists and clinicians were
constrained by donor availability.
Gene therapy changed all of that by
facilitating cell manipulation. Through
recombinant DNA, it was now possible
to restore lost functions, introduce
new functions, or modulate existing
functions. It also became possible to
make adjustments to existing cellular
components, whether receptors or
paracrine molecules, to increase affinity
and efficacy. Not only could a patient’s
cells be extracted and manipulated,
but improved cell culture techniques
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also enabled these recombinant cells
to be expanded and mass produced ex
vivo for continuous use. Theoretically,
this could be done to an infinite extent,
thus alleviating much of the reliance on
finding the right donor.2
A Happy Synergy
Today, scientists use cell and gene ther-
apy to discover new ways to combat
countless different conditions, ranging
from hereditary genetic disorders to
cancer and cardiovascular disease. As of
2020, before the interruptions caused
by the COVID-19 pandemic, there were
roughly ~1,300 ongoing cell and gene
therapy clinical trials globally, a number
that increases year-by-year.3 This trend
is likely to continue for the foreseeable
future, as the discovery of novel gene
editing technologies such as CRISPR
only contributes to gene and cell ther-
apy’s potential.
See references on page 7
3
 GATHERING MOMENTUM:
THE CURRENT STATE
OF CELL AND GENE
THERAPY
T
he fields of cell and gene
therapy have existed for a
long time, first as indepen-
dent areas of study, and
more recently, combined.
Throughout this history, a number of
key techniques have facilitated cell and
gene therapy strategies in the labora-
tory and the clinic. Today, as technol-
ogies such as CRISPR make gene edit-
ing and manipulation easier and more
accessible, cell and gene therapy is well
poised to help shape therapeutics in the
21st century.
Learning New Tricks: Improving
Viral Vectors
The natural properties of viruses make
them well suited for gene delivery.
However, viruses can elicit immune
responses, produce off-target activity,
and result in insertional mutagenesis.
This has resulted in unwanted clinical
outcomes, forcing the scientific com-
munity to exercise caution and design
better and safer vectors.1,2 A wide range
of viruses are used as vectors for gene
therapy, both in the lab and in the clinic.
The most popular of these are adenovi-
ruses, adeno-associated viruses (AAVs),
and lentiviruses.1,2
Adenoviruses were the first viral
vectors approved for clinical use. They
are popular because they possess broad
tissue tropism, but have limited clini-
cal applicability because they are poten-
tially immunogenic and can be neutral-
ized by immune memory. More recently,
the advent of AAVs has largely sup-
planted the use of adenoviruses for both
research and clinical purposes. AAVs
also have broad tropism, but are much
THE PAST, PRESENT, AND FUTURE OF CELL AND GENE THERAPY
less immunogenic.2 This lack of immu-
nogenicity has made it the most popu-
lar choice when in vivo gene therapy is
required, as well as for the majority of
laboratory work. 3
Both adenoviruses and A AVs do
not integrate their payloads into the
host genome, meaning that any genetic
manipulation will not pass onto sub-
sequent generations. Retroviruses are
the preferred vectors when stable inte-
gration is required. 2,3 In particular,
lentiviruses, a genus of retroviruses,
are popular because they can infect
both dividing and non-dividing cells.2
Because retroviruses and lentiviruses
make permanent alterations to the host
genome, there is a risk of mutagenesis
or oncogene activation. To mitigate this
risk, researchers can direct the site of
integration using elements such as zinc
finger nucleases, or use self-inactivating
or non-oncogenic strains.2
Enter CRISPR
The problems associated with viral vec-
tors led to the pursuit of alternative
gene editing approaches. In particular,
researchers have sought to devise ways
to directly modulate gene sequences in
situ.4 The CRISPR/Cas system offers this
possibility. Originally discovered in bac-
teria, this system endogenously cleaves
DNA or RNA at specific sites defined
by single-stranded guide RNAs.5 This
system can be used to inactivate genes
by excising nucleic acid segments, acti-
vate genes by attaching transcriptional
promoters to “dead” Cas proteins lack-
ing endonuclease activity, and modify
sequences with the aid of exogenous oli-
gonucleotides.4,6  The CRISPR/Cas sys-
The Scientist 2021 | the-scientist.com
tem is also flexible; targeting new sites
simply requires altering guide RNA
sequences. Moreover, CRISPR/Cas is
amenable to multiplexing by using mul-
tiple guide RNAs. Most importantly, the
relative novelty of the CRISPR/Cas sys-
tem, combined with the vast diversity
of natural CRISPR systems, means that
many new CRISPR-based gene editing
technologies and strategies are likely to
emerge in the future.7
Bench and Bedside
Cell and gene therapy techniques have
benefitted both the laboratory and the
clinic. Recombinant DNA is critical to
the pharmaceutical industry, as it enables
biologics production and the development
of compound screening and drug test-
ing assays. Viral vectors and CRISPR/
Cas have both been used to create induc-
ible animal models of disease, giving
researchers a better look at pathogenic
mechanisms as they unfold. Similarly, sci-
entists use genetically manipulated cells
for a host of applications, especially in tis-
sue regeneration and stem cell research.
Perhaps the most famous synergy of
cell and gene therapy to date is chime-
ric antigen receptor (CAR) T cell therapy.
Here, gene sequences encoding custom
engineered T cell receptors are incorpo-
rated into T cells ex vivo. The newly cre-
ated CAR T cells are then expanded and
introduced to patients. CAR T cells have
produced significant clinical responses
against certain leukemias and lympho-
mas, and scientists continue to develop
improved T cell receptors to combat anti-
gen escape and cytokine-related toxicity.8
See references on page 7
4
 TOP SPEED: THE
FUTURE OF CELL
AND GENE THERAPY
A
fter decades of up and downs,
2016 marked the first clin-
ically approved cell and
gene therapies. These initial
breakthroughs were quickly
followed by a torrent of additional successes,
including oligonucleotide, cell, and gene-
based therapies treating a diverse range of
clinical indications and tissue targets. These
successes can also be extrapolated to other
conditions and patient populations.1 While
obstacles still remain, cell and gene therapy
is well poised to enter the mainstream.
Improving the Known, Revealing
the Unknown
Cell and gene therapy has taken major
steps in the last five years, and the advent
of next-generation technologies is a major
contributing factor. For example, research-
ers are engineering new viral vectors to
evade immune system neutralization and
investigating alternative vector options
such as nanoparticles. They are also taking
advantage of technologies such as CRISPR
to investigate the therapeutic potential of
in situ gene manipulation. Although CRIS-
PR-based gene editing is still in its infancy,
the scientific community is already look-
ing beyond. Techniques such as base edit-
ing and prime editing permit sequence
alterations without DNA breaks or endog-
enous repair pathways, while RNA-target-
ing and epigenome editing induce reversible
changes to gene expression.1
In addition to these technologi-
cal advancements, cell and gene therapy
has been greatly aided by an exponential
increase in knowledge. Functional genom-
ics, powered by large-scale next-generation
sequencing and data analysis, is reshap-
ing the understanding of how the human
THE PAST, PRESENT, AND FUTURE OF CELL AND GENE THERAPY
genome is modulated and regulated. Pres-
ently, the functions of as much as 30% of
the genes in the human genome remain
unknown.1 As these genes are investi-
gated, new areas of disease biology and
classes of therapeutic targets will arise,
enabling human disease to be dissected and
addressed from new perspectives.
Scaling Up with Progress
With this boom in interest, cell and gene
therapy now faces the challenge of scaling up.
Laboratories need greater flexibility, higher
throughputs, and rigorous consistency.
These adjustments are necessary to meet
future medical demands, as more cell and
gene therapy products enter the clinic, and
address future research questions brought
forth as scientists access larger volumes of
genomic and cytomic data at greater depths.
Many current manufacturing processes
for cell and gene therapies are largely man-
ual, relying on operator experience and
judgement. To complicate matters further,
cell and gene therapies often work with
patient or donor cells, which are innately
heterogeneous and complex. As such, minor
changes in the operator or the environment
can result in considerable batch-to-batch
variation.2 Automation is therefore neces-
sary to provide more control over biopro-
cess consistency and to allow faster and
more efficient process optimization. To this
end, the latest automation platforms cou-
ple mechanization with digitization, pro-
viding a fully automated product assembly
line equipped with process validation and
monitoring sensors.2 In the future, these
platforms could form large parts of fully
insular production environments, thereby
removing the need for clean room infra-
structure. They could also integrate with
The Scientist 2021 | the-scientist.com
artificial intelligence and machine learning
to highlight potential trouble steps in the
manufacturing process and reveal patterns
and correlations between certain biological
characteristics and process outcomes.2
The Importance of Quality
Quality control plays a large role in cell and
gene therapy processes to ensure product
safety and data viability. As cell and gene
therapy workflows attain higher sensitiv-
ities and throughputs, quality control pro-
cesses must keep pace through technolo-
gies like Droplet Digital PCR (ddPCR). In
contrast to qPCR—commonly used to eval-
uate viral titers—ddPCR partitions sam-
ples into tens of thousands of individual
partitions. By amplifying these partitions
separately, ddPCR provides better preci-
sion and reduces data skew risks associated
with amplification bias and miscalibrated
reference standards.3 In addition to calcu-
lating viral titer, ddPCR has also been used
to count cells and measure absolute DNA
copy numbers during long-term storage.4,5
Toward a Personalized Future
As scientific and medical communities
continue to aim towards precision med-
icine, gene and cell therapy is tremen-
dously exciting. Technological leaps over
the past decade have provided unparal-
leled control over nucleic acid delivery,
immune modulation, and human genome
manipulation. These technologies have
tremendously improved the capabilities of
cell and gene therapies, and revealed that
cell and gene therapy is still very much in
its nascent stages.
See references on page 7
5
Gene Manipulation 101
Gene Manipulation

Viral Vectors
CRISPR
Viral vectors carry exogenous
The CRISPR/Cas system allows nucleic acid payloads to cells
targeted in situ gene manipula- for incorporation. Mutagenesis,
tion. Its flexibility and versa- immunogenicity, and toxicity
tility offers broad applicative pose concerns, but viral vectors
potential. are still broadly used in research,
industry, and therapeutics.

Verifying Gene Expression

Tagging qPCR/ddPCR
Attaching reporter genes such Scientists can measure transgene
as GFP or LacZ to sequences levels directly using PCR. qPCR is
of interest allows researchers to popular but is susceptible to am-
quickly confirm transgene uptake plification or standard calibration
and incorporation by measuring biases. Newer technologies like
produced signal. Droplet Digital PCR offer better
precision and accuracy.

Protein-Level Quality Control

Western Blotting ELISA
Transgene incorporation does Transgenes are sometimes
not always translate to protein introduced to drive cellular para-
production. Western blotting crine activity. Techniques like
evaluates the presence of the ELISA are useful for determining
protein encoded by the gene whether proteins of interest are
of interest. being secreted by transfected/
transduced cells.

End Goals

Gain/Loss of Function Novel Engineering Expression Modulation

Reversing gain or loss of function mu-
tations can be instrumental in treating
hereditary genetic disorders. It can also
be used to counterbalance pathogenic
mechanisms.
Gene sequence manipulation can alter
protein structures to add new capabilities
or modify existing properties. CAR T cell
receptors are one example where gene
editing confers improved functionality.
Many pathogenic mechanisms stem from
gene over- or underexpression, resulting
in a loss of balance. Strategies involv-
ing gene expression modulation seek
to restore homeostatic physiological
mechanisms.
References

Article 1: Starting Blocks: An Introduction Article 3: Top Speed: the Future of Cell
to Cell and Gene Therapy and Gene Therapy
1 ) T. Wirth et al., “History of gene therapy,” 1 ) K. Bulaklak, C.A. Gersbach, “The once
Gene, 525(2):162-69, 2013. and future gene therapy,” Nat Commun,
2 ) H. Hentze et al., “Cell therapy and the 11(1):5820, 2020.
safety of embryonic stem cell-derived 2 ) P. Moutsatsou et al., “Automation in cell
grafts,” Trends Biotechnol, 25(1):24-32, and gene therapy manufacturing: from
2007. past to future,” Biotechnol Lett, 41:1245-
3 ) “Cell and gene therapy clinical trials – 53, 2019.
Clinical trials database,” Cell and Gene 3 ) B. Furuta-Hanawa et al., Two-dimensional
Therapy Catapult, accessed June 3, 2021. droplet digital PCR as a tool for titration
https://ct.catapult.org.uk/clinical-trials- and integrity evaluation of recombinant
database. adeno-associated viral vectors,” Hum Gene
Ther Methods, 30(4):127-36, 2019.
Article 2: Gathering Momentum: the 4 ) B. Fehse et al., “Digital PCR assays for
Current State of Cell and Gene Therapy precise quantification of CD19-CAR-T
cells after treatment with axicabtagene
1 ) K. Lundstrom. “Viral vectors in gene
ciloleucel,” Mol Ther Methods Clin Dev,
therapy,” Diseases, 6(2):42, 2018.
16:172-78, 2020.
2 ) R. Goswami et al., “Gene therapy leaves a
5 ) Y. Wang et al., “Monitoring long-term
vicious cycle,” Front Oncol, 9:297, 2019.
DNA storage via absolute copy number
3 ) “Cell and gene therapy clinical trials –
quantification by ddPCR,” Anal Biochem,
Clinical trials database,” Cell and Gene
583:113363, 2019.
Therapy Catapult, accessed June 3,
2021. https://ct.catapult.org.uk/clinical-
trials-database.
4 ) F. Uddin et al., “CRISPR gene therapy:
applications, limitations, and implications
for the future,” Front Oncol, 10:1387, 2020.
5 ) H. Li et al., “Applications of genome editing
technology in the targeted therapy of
human diseases: mechanisms, advances
and prospects,” Signal Transduct Target
Ther, 5(1):1, 2020.
6 ) P. Perez-Pinera et al., “RNA-guided
gene activation by CRISPR-Cas9-based
transcription factors,” Nat Methods,
10(10):973-76, 2013.
7 ) M.L. Maeder, C.A. Gersbach, “Genome-
editing technologies for gene and cell
therapy,” Mol Ther, 24(3):430-46, 2016.
8 ) R.C. Sterner, R.M. Sterner, “CAR-T cell
therapy: current limitations and potential
strategies,” Blood Cancer J, 11:69, 2021.

THE PAST, PRESENT, AND FUTURE OF CELL AND GENE THERAPY The Scientist 2021 | the-scientist.com 7
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