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K. Gadamasetti
critical process in the lifecycle of biotech products. It enables the production of
clinical material to test a hypothesis developed in the drug discovery environment,
expansion of clinical supplies, and establishment of a robust platform for commercial production. It can also provide exibility in the product lifecycle by adding
additional manufacturing nodes to supplement supply or provide business continuity.
1.5.10 Emerging Trends andFuture ofBiopharmaceuticals
The last chapter by Woods and Gierach from Department of Chemical and
Biomolecular Engineering of The Ohio State University entitled, “Emerging
Biopharmaceutical Technologies and Trends” introduces a few of the high-prole
changes taking place in the industry, both in terms of new treatments and modalities
and new manufacturing approaches.
1.6 Future ofBiologics
Synthetic biology leading to biologics is here to stay and thrive for decades ahead.
It will transform how we grow food, what we eat, and where we source materials
and medicines from. Synthetic biology enabled in advancing metabolic engineering, directed evolution-automated strain engineering, metagenomic discovery, gene
circuit design, and genome editing. Products from synthetic biology are rapidly
growing and by 2030, it is highly likely that you will have eaten, worn, used, or been
treated with one [11].
References
1. Schrödinger E (1944) What is life? The physical aspect of the living cell, 1st edn. Cambridge
University Press, Cambridge. Schrödinger’s contribution to the eld of molecular biology from
the perspective of quantum physics. ‘Aperiodic crystal’ is currently called the protein structure
2. Banting FG, Best CH, Collip JB, Campbell WR, Fletcher AA (1922) Pancreatic extracts in the
treatment of diabetes mellitus. Can Med Assoc J 12(3):141–146. https://www.ncbi.nlm.nih.
gov/pmc/articles/PMC1524425/
3. Insulin is a biologic—What does that mean? https://www.diabetes.org/blog/insulin- now- biologic-
what- does- mean
4. The readers are encouraged to follow the citations 4a and 4c below especially, if new to this
subject material of genetics:
4a. Genetics home reference: help me understand genetics. https://ghr.nlm.nih.gov/ and https://
medlineplus.gov/genetics/
4b. The health conditions in https://medlineplus.gov/genetics/condition/. Lamins A and C are
structural proteins called intermediate lament proteins. Intermediate laments provide stability and strength to cells
1 Bioprocessing, Bioengineering and Process Chemistry in the Biopharmaceutical…
4c. https://www.genome.gov/genetics- glossary/Point- Mutation
4d. Mayo Clinic. https://www.mayoclinic.org/diseases- conditions/progeria/diagnosis- treatment/
drc- 20356043
4e. Aguado J, d’Adda di Fagagna F etal (2019) Inhibition of DNA damage response at telo-
meres improves the detrimental phenotypes of Hutchinson–Gilford Progeria Syndrome. Nat
Commun 10:4990. https://doi.org/10.1038/s41467- 019- 13018- 3
4f. Rees HA, Leu DR (2018) Base editing: chemistry on the genome and the transcriptome in liv-
ing cells. Nat Rev Genet 19:770–788
5. Clancy S (2008) Genetic mutation. Nat Educ 1(1):187. https://www.nature.com/scitable/
topicpage/genetic-mutation-441/
6a. Benner SL etal (2019) Hachimoji DNA and RNA: a genetic system with eight building blocks.
Science 363(6429):884–887 and the references therein
6b. Romesberg FE et al (2019) Eight-letter DNA. Biochem 58(22):2581–2583 and references
therein; expanded genetic alphabet, Romesberg FL and coworkers, Acc Chem Res, 2018, 51,
2:394–403. https://pubs.acs.org/doi/full/10.1021/acs.accounts.7b00403, Angew Chem Int Ed
Engl 2015, 54(41):11930–11944
6c. Hirao I etal (2021) Cognate base-pair selectivity of hydrophobic unnatural bases in DNA liga-
tion by T4 DNA ligase. Biopolymers 112:e23407. https://doi.org/10.1002/bip.23407 and references therein; Genetic alphabet expansion transcription generating functional RNA molecules
containing a ve-letter alphabet including modied unnatural and natural base nucleotides by
thermostable T7 RNA polymerase variants, Kimoto M, Meyer AJ, Hirao I, and Ellington AD
(2017) Chem Commun, 53:12309–12312 and references therein
7a. Sickle cell disease, Gregory J.Kato and co-authors, Nat. Reviews, 2018, 4, Article No. 18010,
1; Ribeil J-A (2017) Gene therapy in a patient with sickle cell disease. New Engl J Med
376:848–855. Mayo Clinic Patient Care and Health Information. https://www.mayoclinic.org/
diseases- conditions/sickle- cell- anemia/diagnosis- treatment/
7b. A CRISPR Focus into the Attitudes and Beliefs of Sickle Cell Patients, Parents, and Providers
Towards Gene Editing, Vence L.Bonham and co-workers, NIH, National Human Genome Res
Instt. 2018
7c. Gabriel Salinas Cisneros and Swee L.Thein, Front Physiol, 2020, 11, Article 435;Genome
Editing Clinical Trials for Sickle Cell Disease, Stacy Desine and co-authors, AJOB, Empir
Bioeth, 2020, 11(4), 195–207
7d. Current and Future Therapeutics for Treating Patients with Sickle Cell Disease by Fortenberry
and co-authors, Cells 2024, 13, 848. https://doi.org/10.3390/cells13100848
8a. Hart SL etal (2020) New approaches to genetic therapies of cystic brosis. J Cystic Fibrosis
19:S54–S59. https://hopkinscf.org/knowledge/cftr/
8b. https://www.mayoclinic.org/diseases- conditions/cystic- fibrosis/diagnosis- treatment/
drc- 20353706
8c. Dechecchi MC etal (2018) Molecular basis of cystic brosis: from bench to bedside. Ann
Transl Med 6(17):334 and references therein
9. Progeria. https://www.mayoclinic.org/diseases- conditions/progeria/symptoms- causes/
syc- 20356038#:~:text=Overview,generally%20appear%20normal%20at%20birth
10. 50 of 2021’s best selling drugs, Brian Buntz, Drug Disc Dev, 29, 2022. https://www.drugdis-
coverytrends.com/50- of- 2021s- best- selling- pharmaceuticals/
11. Voigt CA (2020) Synthetic biology 2020–2030: six commercially-available products that are
changing our world. Nat Commun 11:1–6
12. Protein metabolism, Lehninger, principles of biochemistry, Chapter 27, ISBN-13:
978-1-4641-2611-6
13. Hanson RL (2008) Process chemistry in the pharmaceutical industry.: Gadamasetti K,
Braish T (eds) Challenges in an ever-changing climate. CRC Press, Taylor & Francis Group,
pp279–294
21
Part II
Synthetic Biology
Chapter 2
Synthetic Biology inDrug Development
andBeyond
AaronK.Sato andStephenRife
Abstract In its simplest denition, synthetic biology is the creation of new biologi-
cal entities for useful purposes. By manipulating an organism’s genome, synthetic
biologists can produce novel proteins for a wide range of applications, from the
biosynthesis of industrial chemicals to the discovery and optimization of biotherapeutics. The eld of synthetic biology has experienced a renaissance in recent years
as technological advances have lowered the barrier to entry and increased the potential for innovation. Principal among these advances has been the development of
highly precise and large-scale DNA synthesis platforms.
The synthesis of genetic material is a non-trivial, yet integral component of synthetic biology. After all, behind every novel protein is a novel DNA sequence.
Synthetic oligonucleotides are also irreplaceable components in CRISPR (clustered
regularly interspersed palindromic repeats) gene-editing, DNA sequencing, and the
myriad tools that enable researchers to manipulate and interrogate genomes.
Historically, oligonucleotide synthesis was a slow, error-prone process that
severely limited its usefulness beyond niche studies. However, the advent of phosphoramidite chemistry and solid-phase synthesis marked an inection point after
which the scale, efciency, and precision of DNA synthesis markedly increased.
And with this increase came an ever-growing list of applications for synthetic
biology.
The eld of synthetic biology is on a trajectory to play a pivotal role in addressing many of the world’s most challenging and complex problems. Projects are
underway to develop and apply engineered organisms in bioremediation, helping to
clean polluted ecosystems. Crops, engineered to resist harsh weather conditions,
have long been sought as a means to reduce starvation in drought-stricken environments. And with the ability to rapidly design and build DNA libraries, drug developers will be better equipped to discover and optimize novel therapeutic modalities.
A. K. Sato (*) · S. Rife
Twist Bioscience, South San Francisco, CA, USA
e-mail: asato@twistbioscience.com
K. Gadamasetti, S. A. Kolodziej (eds.), Bioprocessing, Bioengineering
and Process Chemistry in the Biopharmaceutical Industry,
https://doi.org/10.1007/978-3-031-62007-2_2
25© The Author(s), under exclusive license to Springer Nature Switzerland AG 2024
26
A. K. Sato and S. Rife
Perhaps the most salient example of synthetic biology’s ability to advance therapeutic development is the rapid expansion of cell therapies to include chimeric antigen receptor (CAR)-T and CAR-NK (natural killer) cells. These highly engineered
cells sit at the vanguard of clinical oncology and, as the tools of synthetic biology
continue to evolve, will likely play an ever-larger role in patient care.
In the following sections, we provide a brief overview of synthetic biology and
how this nascent eld is catalyzing rapid development of novel biotherapeutics.
Keywords Synthetic biology · Genetic circuits · Chassis organism · Hybridoma
cells · Phage display
2.1 What Is Synthetic Biology?
In the throes of the industrial revolution, Mary Shelley sat down to write the nowfamous story of Frankenstein. Having listened to galvanists argue over the mechanical and potentially electric nature of life, she was inspired by the possibility that
“perhaps the component parts of a creature might be manufactured, brought together,
and endued with vital warmth” [1]. Given the right knowledge set and tools, Shelley
and many scientists believed that someday we may be able to engineer novel forms
of life. More than 150years later, the eld of synthetic biology began.
Synthetic biology describes the general pursuit of solutions to the world’s problems through the deliberate engineering of biological systems. It is a distant echo
from what Shelley and her contemporaries envisioned, but synthetic biology retains
the central notion that living organisms are a complex assemblage of parts whose
activity can be both predicted and manipulated. As such, synthetic biology can modify—or in some cases, create—biological systems that perform specic, predictable
functions.
For example, genes can be conceptualized as discrete “parts” that reliably produce proteins when in the right environment. It stands to reason that parts can be
swapped and that inserting a specic gene into an organism’s genome will enable
that organism to start producing the corresponding protein. Such was the reasoning
when researchers from UC Berkeley modied the Escherichia coli genome to contain a myriad of foreign genes sourced from plants, yeast, and bacteria [2]. The
result was a novel bacterium whose synthetic genome gave it the power to convert a
common metabolic byproduct (acetyl-CoA) into the antimalarial therapeutic artemisinin. In subsequent years, approximately 39 million treatments of artemisinin
were produced through synthetic means [3].
As demonstrated in the example of artemisinin, synthetic organisms can be engineered to produce complex chemicals that may be completely novel, or else only
exist in specic ecological niches. Artemisinin is naturally produced by sweet
wormwood (also known as qinghao, Artemisia annua) in very low quantities [4, 5].
2 Synthetic Biology inDrug Development andBeyond
27
Extraction of the compound is a costly process and requires large quantities of the
host plant. By producing artemisinin with a synthetic organism, researchers gain a
stable therapeutic source that may increase the scale of production while reducing
the process’ costs and environmental impacts [5].
The potential applications of synthetic biology extend well beyond drug manufacturing. Already organisms have been engineered to improve crop yields, synthesize complex materials, and produce commercial chemicals on an industrial scale
[6]. Yet, the eld is still in its infancy, having only begun in earnest at the turn of the
twenty-rst century [7].
In these nascent stages, the eld must not only grapple with technological and
scientic challenges but also those concerning philosophical and ethical questions.
As in the days of Mary Shelley, the notion that life can be re-engineered or otherwise articially altered raises questions about what constitutes life and what boundaries should be placed on the engineering of living organisms. These questions drive
much of Shelley’s novel as well as modern discourse about the potential—both
good and bad—of synthetic biology [8].
As these questions continue to be explored, the eld of synthetic biology continues to mature and expand in its applications. While the full potential of synthetic
biology is yet to be realized, it is clear that this eld will dramatically inuence—
and indeed has already inuenced—the drug discovery and development industry.
In this chapter, we explore synthetic biology and its ongoing inuence in modern
drug development. We begin by outlining the basic tools used in synthetic biology.
We then provide a high-level overview of several ways in which these tools are used
to advance drug discovery and development, from CRISPR screening to chimeric
antigen receptor (CAR)-T cell therapies. While this is not an exhaustive overview,
we hope that the reader will nd it a useful introduction to the fast-expanding application of synthetic biology in drug development.
2.2 A Brief Overview ofSynthetic Biology Tools
At this point, it is important to briey review the tools that are commonly employed
in synthetic biology, namely, synthetic DNA, gene assembly methods, gene circuits,
and chassis organisms.
Synthetic DNA
Solid-phase DNA synthesis has had a transformative impact on the molecular sciences, including synthetic biology. This chapter will include a brief discussion of
DNA synthesis technology, but for an in-depth exploration of synthesis chemistry,
readers may nd a recent review from Hughes et al., in Cold Spring Harbor
Perspectives in Biology, helpful [9]. Modern advances in automation, solid-phase
synthesis, and phosphoramidite chemistry have made it possible to precisely synthesize millions of oligonucleotides in a matter of days.
28
A. K. Sato and S. Rife
Synthesis companies can now reliably produce large-scale libraries consisting of
distinct oligonucleotides up to 300 nucleotides in length. The added length and
scale of synthesis has enabled rapid gene assembly, genome-wide single-cell
CRISPR screening, and a multitude of other applications. Scale, precision, and uniformity are all important factors that will be discussed later in this chapter, but the
important point is that technological advances have made it possible to routinely
synthesize genetic sequences that can then serve as modular parts for synthetic biology applications.
Gene Assembly
Modularity is a core feature in synthetic biology as it allows for parts to be easily
swapped when needed. Swapping segments of DNA with precision became possible in the 1970s with the advent of restriction enzyme digestion and ligation-based
assembly methods [10]. Though useful, these original methods suffered from low
delity and the need for unique restriction enzyme recognition sites—greatly limiting the scale and modularity of their application. Contemporary assembly methods
include Biobrick Assembly, Golden-Gate Cloning, and Gibson Cloning [10, 11].
These methods make use of DNA scaring, sticky end ligation, and other techniques
to achieve high-delity gene assembly. While none are perfect, these methods have
been optimized to allow for many gene parts to be rapidly and reliably assembled
into larger gene clusters. When these gene clusters interact with one another in a
predictable and controlled manner, they may be referred to as circuits.
Genetic Circuits
Genetic circuits are biological networks designed to carry out logic functions akin
to computational circuits. A more formal denition may describe them as interlinked transcriptional and posttranscriptional networks whose activity regulates
molecular processes, such as gene expression. Genetic circuits can be designed to
express molecules—such as reporters or specic enzymes—only when a specic
stimulus is present. In the case of circuits following AND logic, the desired molecule is only expressed when multiple conditions are true, such as the presence of a
synthetic transcription factor and an accessible target promoter [12–14].
Alternatively, an engineered circuit may express different proteins depending on the
cell’s environment, as in the case of NOR/NAND circuits [14].
The ability to program precise, conditional gene expression opens the door to
many possible applications in drug development and target validation. Many natural
therapeutics are built through a series of enzymatic relays that may involve negative
and positive feedback loops and may require compartmentalization. Genetic circuits can be built to replicate these natural biosynthetic pathways in yeast, bacteria,
and other chassis organisms, enabling the construction of complex chemicals en
masse [12, 13].
Chasses
Finally, chassis organisms are the workhorses of synthetic biology. A chassis in
synthetic biology is an organism that can host synthetic parts (genes, gene clusters,
etc.) and provides the machinery for those parts to function, such as transcriptional
2 Synthetic Biology inDrug Development andBeyond
and translational proteins. Two of the most commonly used chasses are Escherichia
coli and Saccharomyces cerevisiae, primarily because these organisms have been
extensively characterized and used in molecular research. Their genomes are well
described and both tools and methods are already established for efcient engineering of their genomes. As such, these organisms can readily be engineered to host
and express new genes and gene circuits in a laboratory environment [15–17].
Together, DNA synthesis and gene assembly technology, genetic circuits, and
chassis organisms represent critical components in a synthetic biologist’s toolkit.
These enable the synthesis of novel DNA sequences, assembly of novel genes and
gene clusters, and culturing of novel genetic sequences in a controlled laboratory
setting—all of which are employed in the discovery and development of new
therapeutics.
29
2.3 Synthetic Biology inDrug Discovery andDevelopment
The synthesis of artemisinin is the most commonly cited example of how synthetic
biology can be applied to drug discovery and development. And while it is an example worth delving into deeper, it is only one of many varied ways that synthetic
biology has been advancing therapeutic development. Below, we provide select
examples of how synthetic biology is currently reshaping this space. An exhaustive
overview would likely warrant its own book. Therefore, we have selected examples
to demonstrate both the core principles of synthetic biology and the breadth of its
application. There are undoubtedly many more applications that are noteworthy but
have been nonetheless excluded from this discussion. We encourage readers to follow their interest to the many citations provided throughout this text for further
reading.
2.3.1 Gene Editing fortheIdentication ofDrug Targets
The term synthetic biology often carries with it an air of artisanal craft, as though
the application of this eld were limited to niche projects. But as this chapter will
emphasize, such a connotation is far from the truth. Perhaps the best example of this
is the widespread application of CRISPR-Cas gene editing technology.
Multiple species of bacteria and archaea contain segments of DNA known as
clustered regularly interspersed palindromic repeats, or CRISPR sequences. These
sequences serve as memory centers wherein DNA that has been harvested from
invading viruses can be stored. When these sequences are transcribed, they produce
spacer RNA (spacerRNA) that isprocessed into a shorter crisprRNA (crRNA) that
is complementary to a specic segment of viral DNA.When crRNA is joined to
both a trans-activating CRISPR RNA (tracerRNA) and a CRISPR-associated (Cas)
nuclease, they form a CRISPR-Cas complex that functions as an adaptive immune