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FIGURE 27.6 Basic components of a cationic lipid. (a) hydrophobic lipid group, (b) linker group, and (c) cationic headgroup.
Pharmaceutical Dosage Forms and Drug Delivery
fuse with the lysosomal cell membrane as the pH becomes acidic, thus disrupting the lysosome and
releasing its cargo. Thus, the plasmid DNA escapes into the cytoplasm without getting degraded within
the lysosome.
ture of the cationic lipid N[1- (2,3- dioleyloxy)propyl]- N,N,N- trimethylammonium chloride (DOTMA)
and the colipid dioleoyl phosphatidylethanolamine (DOPE). Cationic lipids interact electrostatically with
or neutral colipid, such as DOPE or cholesterol, respectively, to form liposomes or micelles. The cationic lipid and colipid are mixed together in chloroform, which is then evaporated to dryness. Water is
As shown in , the general structure of a cationic lipid has three parts: (1) a hydrophobic
lipid anchor group, which helps in forming liposomes (or micellar structures) and can interact with cell
headgroup, which interacts with the plasmid, leading to its conden-
linker group that connects the lipid anchor with the charged headgroup. The net charge
-
ciency in vitro
the physicochemical properties of liposome/ plasmid complexes.
27.7.3.2 Peptide- Based Gene Delivery
- lysine)
(PLL), histones, protamine, or poly(
then complexed to plasmids via electrostatic interaction. The resulting complexes retain their ability to
into the cells. Receptor ligands currently being investigated include glycoproteins, transferrin, polymeric
-
tein A and B, mucin, and the c- kit receptor.
method of complexation, the molecular weights of both polycations and plasmids and the number of
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ligand residues bound per polycation molecule. To avoid high cytotoxicity, molecular heterogeneity,
and possible immunogenicity of PLL and polyethylenimine (PEI), molecularly homogenous lysine and
arginine- rich peptide- based gene delivery systems are being investigated. Peptides with moieties that provide cooperative hydrophobic behavior of the alkyl chains of cationic lipids would improve the stability
lytic properties, with little activity at pH 7 but greater than or equal to a 100- fold increase in transfection
27.7.3.3 Polymer- Based Gene Delivery
Polymeric biomaterials can be tailored to interact more with cellular and protein levels to achieve high
(3) polymer/ cell matrix, (4) functional biodegradable polymers, and (5) polymeric gene carriers. These
as polyvinylpyrrolidone (PVP) and pluronics, can also be used to deliver nucleic acids to muscles and
tumors. These polymer- based DNA formulations are hyperosmotic and result in an improved dispersion
of plasmids through the extracellular matrix of solid tissues, such as muscles or solid tumors, possibly by
protecting plasmids from nuclease degradation, dispersing plasmids in the muscle, and facilitating their
of DNA condensation, targeting, cellular uptake, intracellular release, and bioactivity.
Review Questions
27.1 The term gene therapy refers to a method
A
therapeutic proteins
B For the treatment of genetic as well as acquired or chronic diseases
C Which allows the production of therapeutic protein or inhibition of abnormal protein production
D Which allows somatic or germ- line cells to produce therapeutic/ reporter proteins
E All of the above
27.2 Gene therapy has great potential because
A It can control the intracellular production of a gene product in response to a disease
B
C It can deliver sustained therapeutic protein levels over a prolonged period
D
E All of the above
27.3
27.4
27.5
Describe the essential features of a gene expression system.
27.7
27.8 The main challenge(s) of mRNA- based therapy is
A
B Stability
C
D immunogenicity
E all of the above
27.9
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FURTHER READINGS
Pharm Res
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Advanced siRNA Delivery
Crooke S.T. (2004) Progress in antisense technology. Annu Rev Med. 55
Pharmacol Rev 58: 32– 45.
Li F. and Mahato R.I. (Eds.) (2015) miRNAs as targets for cancer treatment: Therapeutics design and delivery.
Adv Drug Del Rev 8: 1– 198.
Mahato R.I. (Ed.) (2005) Biomaterials for Delivery and Targeting of Proteins and Nucleic Acids, Boca Raton,
FL: Taylor & Francis Group.
oligodeoxynucleotides and small interfering RNA. Expert Opin Drug Deliv 2: 3– 28.
Pharmaceutical Perspectives of Nucleic Acid- based Therapeutics,
London: Taylor & Francis Group.
Mahato R.I., Smith L.C., and Rolland A. (1999) Pharmaceutical perspectives of nonviral gene therapy. Adv
Genet 41
Smith A.E. (1995) Viral vectors in gene therapy. Annu Rev Microbiol 49: 807– 838.
Pharmaceutical Dosage Forms and Drug Delivery
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28
Genome Editing for Therapeutic Applications
This chapter will provide a brief introduction to the basic concepts of genome editing, different tools commonly used for gene editing, the role of gene editing in drug discovery and therapeutics, and challenges.
On completion of this chapter, the students should be able to
1.
2. Describe the basic techniques of genome editing.
3.
4. Describe different challenges associated with gene editing.
28.1 Introduction
Gene therapy is the replacement of defective genes or the insertion of new genes to treat illness or
increase disease resistance. Genome editing is a fundamental component in the domain of gene therapy,
molecular biology has enabled the understanding of the function of genetics in a variety of diseases,
and blindness. Gene altering in somatic cells has the potential to restore normal function in disease
tissues, whereas gene altering in the germline has the potential to eliminate hereditary disorders in future
generations.
sequence. When a double- stranded break (DSB) occurs in DNA, the cell’s own DNA repair processes are
metabolic responses or replication stress or by external events like radiation or chemotherapeutics.
pathways that a cell employs to repair double- strand breaks (DSBs) (Figure 28.1).
IIS restriction endonuclease FokI. In the presence of two sets of ZF domains arranged in the proper
(Figure 28.2
any target DNA sequence and off- target effects.
LEARNING OBJECTIVES
DOI: 10.1201/9781003389378-32
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FIGURE 28.1 Schematic diagram showing the single-strand break (SSB) and double- strand break (DSB) repair pathways
of DNA.
FIGURE 28.2 Different Gene editing methods.
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TALENs sequences originated from pathogenic bacteria in plants and contained 33– 35 amino acids
in length DNA- binding proteins known as TALEs. These TALEs can be engineered to identify a spe-
straightforward protein- DNA code of TALENs enables effortless construction to target virtually any
clustered regularly interspaced short palindromic repeats (CRISPR). The concept of RNA- guided
targeting was taken from bacteria’s natural defense mechanisms against viral infections. Cas9 protein is
used as a nuclease by CRISPR to cleave the DNA. CRISPR/ Cas9 has several important advantages over
other gene editing technologies. For instance, the ZFN or TALEN- based tools require reengineering the
Some of these gene editing approaches have been employed successfully in basic, preclinical, and
clinical contexts in recent years. Basic research might study cellular, molecular, genetic, or immunological systems that affect reproduction, disease development, and therapy responses. For the most part,
somatic cells, like those found in the skin, liver, lungs, and heart, are used in basic research on human
cells. However, some basic research makes use of germline cells like those found in early-stage human
embryos, eggs, and sperm. Research employing somatic cells to alter the genome facilitates the develop-
to their offspring. Applications may involve extracting blood or bone marrow cells from a person, making
genetic alterations (in vitro), and returning them. Somatic genome editing can also be done in vivo by
Germline cell genome editing studies can help understand human development and fertility. Thus,
editing the germline cells of individuals who carry these mutations could allow them to have genetically related children without the risk of passing on these conditions. Thousands of hereditary diseases
are caused by mutations in a single gene, making germline gene editing extremely intriguing. By modifying one or more genes in a genetically related animal like a mouse, scientists may examine how these
human health.
Genome editing is applied in clinical research when traditional medicines are ineffective for treatment.
Several disease conditions, including but not limited to antibacterial infections, blood disorders, cancer,
ocular disorders, and metabolic disorders, are currently undergoing clinical trials employing CRISPR
technology. Recently approved clinical trials included individuals with advanced cancer who have not
responded to standard therapies such as chemotherapy and radiation. Through the use of genome editing,
immune cells from patients are being programmed to target malignancy in this study (Figure 28.3).
questions, ethical issues, and societal implications. Here, we discuss genome editing platform clinical
trials for disease treatment and their implementation issues.
28.2 Breakage and Repair Mechanisms of Genomic DNA
dentally (e.g., by radiation) or purposefully, using proteins called endonucleases (often called nucleases)
that can generate DSBs in DNA. Cells developed intricate signaling networks to detect DNA damage,
orchestrate cell cycle checkpoints, and repair them. Repairing DNA DSB can be accomplished by a variety of methods. However, there are two main categories based on whether a homologous DNA sequence
is used as a template. Homolog recombination (HR) DNA repair uses a homologous DNA sequence as
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FIGURE 28.3 Various applications of genome editing.
Pharmaceutical Dosage Forms and Drug Delivery
a template for DNA synthesis and involves DNA- end processing. In contrast, the non- homologous end
ends of broken DNA.
28.2.1 Homolog Recombination (HR) DNA Repair
The HR DSB repair is carried out when the homologous DNA strand is in proximity so one strand can
be used to repair the other. In eukaryotes, the damaged ends of DNA are degraded by a nuclease protein complex consisting of MRE11- RAD50- NBS1 (MRN). The DNA is left with a single- stranded 3– 5
Subsequently, the protein RAD51 is activated by adenosine triphosphate (ATP) binds to the DNA and
invading strand searches for undamaged homologous sequences in the undamaged sister chromatin by
base pairing in a block of three nucleotides. If the base pair misses the match, the invading DNA looks for
matching, a displacement loop structure is formed by the DNA polymerase using the invading strand as a
template. Following this, the helicase displaces the newly formed invading strand, which base pairs with
the uncoded damaged strand. Next, the second damaged strand anneals to the undamaged complementary
strand for another round of DNA synthesis. Finally, the sister strands dissociate, and DNA ligase seals
the nicks, restoring the repaired helices. HR is a prone free DNA repair mechanism that occurs primarily
during cell proliferation.
28.2.2 Nonhomologous End Joining (NHEJ) Repair
single- stranded tails that are present at the break. The broken end is ligated back together by Ligase IV,
Figure 28.1). Consequently, the duplex is
reconnected, although with the loss of a substantial segment of DNA that had been excised by nucleases.
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Therefore, this mechanism of DNA repair does not reinstate the precise sequence of the DNA and can be
used to generate alterations in the DNA sequence.
removal of DNA sequences of diverse lengths, thereby potentially perturbing the functionality of genes.
Nevertheless, the introduction of a homologous stretch of DNA into the cell as a donor template can result
in enhanced repair accuracy via homology- directed repair (HDR). Furthermore, if the donor template
sequence.
28.3 Genome Editing Techniques
helix. Subsequently, scientists have dedicated several decades to unraveling seven effective methodolo-
TALENs were the three main methods for creating targeted cleavages in DNA using nuclease systems.
28.3.1 Restriction Enzymes
opportunity to introduce new DNA material at the spot where the cut was made. There are mainly three
composed of three types of proteins that function as a single unit. The complex typically consists of one
-
metric or palindromic sequence and cuts within that sequence. A sequence of nucleotides is considered
palindromic if it is identical to the reversal of its complement. ACCTAGGT, for instance, is a palindrome.
The complementary strand will contain the sequence TGGATCCA, which remains unchanged even when
inverted. The monomer and homodimer subunit complex handles four and six recognition sequences,
HindII and HindIII, are preferred over others in molecular biology because they cut right through the
recognition site.
(M) and restriction endonuclease (R), which work together as a single protein complex. Protein M is
-
plex, cleaves downstream of the recognition site, typically 25– 28 bases away.
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molecular biology, such as in DNA mapping, epigenome mapping, and constructing DNA libraries.
28.3.2 Zinc Finger Nucleases
gene- editing technique was imperative given the potential for detrimental off- target effects. In response,
tion to identify and bind to a particular sequence (of amino acid) in the DNA. ZFNs comprise a genetic-
DNA- binding domain can identify a location on DNA consisting of three base pairs. ZFNs act as dimers,
the absence of frameshift mutations.
ment for a three-base pair. Further, target sites with a high guanine content demonstrated greater editing
editing.
human clinical trials, the most advanced of which is in the treatment of HIV/ AIDS, where deletion of
the chemokine receptor 5 (CCR5) HIV coreceptor has the potential to enable the elimination of HIV
following bone- marrow transplantation. Following ZFN- mediated editing, scientists found autologous
CD4+ T- cells were safe to use and had an exciting potential for HIV therapy. Additionally, ZFNs have
and NCT02702115 [in vivo editing MPS1]).
28.3.3 Transcription Activator- Like Effector Nucleases (TALEN)
of transcription activator- like effectors (TALEs), which are tandem arrays of 33– 35 amino acid repeats.
of the DNA in the genome. Basically, the DNA- binding selectivity is determined by a Repeat Variable
Di- residue (RVD) located in the amino acid repeat. A total of over 20 distinct RVD sequences have been
when it is in the dimer form. One crucial factor in the cleavage is the distance between the two binding
sites, which is typically around 15 base pairs. TALENs are regarded as the safest gene- editing method
Further, researchers employed TALENs to treat COLA7A1 dysfunction in patients with epidermolysis
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bullosa, a condition marked by a loss of skin integrity that increases the risk of skin cancer and can result
in potentially deadly skin blisters.
Nevertheless, TALENs have certain drawbacks, including their time- consuming and expensive nature,
as well as certain limits in their design. The design of TALENs necessitated the presence of a thymine
demonstrated advancements in genome editing technology, their extensive implementation was impeded
28.3.4 Meganucleases
Homing endonucleases, also referred to as meganucleases, are proteins that occur naturally in the
genomes of chloroplasts and mitochondria. Meganucleases have very long DNA- binding recognition
sites, up to 40 nucleotides. As a result of their length, it is exceedingly unlikely that natural sites would
be present by chance, even in complex human genomes. Meganucleases that possess a single conserved
LAGLIDADG motif per protein chain form homodimeric proteins that cleave palindromic and nearly
palindromic DNA target sequences, while those that contain two such motifs per protein chain form
larger, pseudo- symmetric monomers that can target completely asymmetric DNA sequences. Two recent
a sequence of interest. Modifying DNA- binding sites and combining meganucleases with TALE DNA-
28.3.5 CRISPR- Cas9
In 2012, scientists discovered a new method of genome editing derived from CRISPR- Cas9. In bacteria,
this adaptive defense mechanism has been around for a very long time, and it helps protect them against
and Emmanuelle Charpentier. Later, Feng Zhang revealed how CRISPR may be used to modify eukary-
technology. Analysis of the genomes of several strains of bacteria led to the discovery of the CRISPR
locus. CRISPR loci’s spacer (nonrepetitive) sections were generated from bacteriophage genomic DNA,
suggesting that CRISPR protected against foreign genetic material.
CRISPR is a complex two- component system comprised of a guide RNA and a Cas9 nuclease with catalytic RuvC and two pairs of conserved histidine and one asparagine (HNH) domains. The Cas9 nuclease
guide RNAs is easier and does not need to be fused with the Cas9 protein, so it is cost- effective, making
it more accessible to scientists. Moreover, algorithms have been constructed to evaluate the probability
of off- target effects, namely if this sequence is present in other locations in the genome. The cleavage
location of Cas9 is determined by the complementary base pairing between single guide RNA (sgRNA)
and the target DNA. sgRNA is a chimera of CRISPR RNA (crRNA) transcribed from the CRISPR locus
and the trans- activating (tracrRNA). Cas9 uses the tracrRNA portion of the guide as a handle, while the
nucleotide base) and, when found, proceeds to unwind the DNA. The PAM sequence is conserved in
PAM on the opposing DNA strand. If the sgRNA is complementary to the DNA, it cleaves the DNA and
creates a DSB at a location that is 3– 5 bases before the PAM. Subsequently, the Cas9 molecule departs
the site and proceeds to search for another PAM site to repeat the process.
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