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FIGURE 27.2 Structures of antisense compounds. (a) antisense oligonucleotide (ODN), (b) peptide nucleic acid (PNA), and
3, methylphosphonate
oligonucleotide.
Pharmaceutical Dosage Forms and Drug Delivery
the production of the protein encoded by the target mRNA. By acting at this earlier stage in the disease-
causing process to prevent the production of a disease- causing protein, antisense drugs have the potential
protein has already been produced. Antisense drugs also have the potential to be much more selective or
through multiple points of interaction at a single binding site. An oligomer of about 15– 20 nucleotides in
length is considered the best because this corresponds to both the appropriate length of a single unique
Figure 27.2
sulfur atom replaces one of the nonbridging oxygen atoms in the phosphate group, produces ODNs that
are relatively resistant to cellular and serum nucleases. Methylphosphonate ODNs have no net charge,
which prevents nuclease digestion but also decreases water solubility (Figure 27.2a).
27.4.2 Triplex- Forming Oligonucleotides
In contrast to antisense ODNs, triplex- forming oligonucleotides (TFOs) inhibit gene transcription by
TFOs to the gene itself presents several advantages compared to antisense ODNs, which are directed to
mRNA. There are only two copies of the targeted gene, whereas there are thousands of copies of mRNA.
Blocking mRNA translation does not prevent the corresponding gene from being transcribed, which continuously repopulates the RNA pool. In contrast, the prevention of gene transcription can reduce mRNA
DNA normally exists in a duplex form (two strands coiled around each other). However, under some
circumstances, DNA can assume triple helix structures. Triplex helix formation may then prevent the
interaction of various transcription factors or physically block the initiation or elongation of the tran-
binding ODNs that can bind to DNA duplex, leading to triple helix formation and prevention of the
transcription process.
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27.4.3 Peptide Nucleic Acids
PNA has a chemical structure similar to DNA and RNA but differs in the composition of its backbone.
DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone
comprises repeating N- (2- aminoethyl)- glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. PNAs are depicted similarly to
the backbone of PNA contains no charged phosphate groups, the binding between PNA/ DNA strands is
stronger than between DNA/ DNA strands due to the lack of electrostatic repulsion. PNAs are resistant to
Therefore, PNAs can be used as antisense medicines similar to ODNs.
27.4.4 Antisense RNA
The antisense mRNA strategy relies on the transfection and subsequent expression of a plasmid vector
whose gene expression cassette carries the cDNA of the gene of interest subcloned into the vector in
an antisense orientation (Figure 27.2c). After transfection (the process of introducing foreign genetic
material into cells) into the cells, the plasmid expresses the antisense mRNA within the cell cytoplasm.
protein synthesis (translation). Hence, antisense mRNA gene medicines require expression vectors and
delivery systems similar to gene therapy medicines (discussed later in this chapter).
27.4.5 MicroRNA
MicroRNAs (miRNAs) are endogenous, noncoding, small (~22 nucleotides) double- stranded RNAs that
participate in gene silencing and posttranscriptional regulation of gene expression. The miRNAs regulate several cellular processes to impact overall outcomes in areas such as cell survival and fat metabolism. Each miRNA has multiple targets and makes global changes in the cellular systems. Changes in
the expression level of miRNAs are associated with phenotypic or performance differentiation among
different cells. For example, high and low titer- producing Chinese hamster ovary (CHO) cell lines have
or gene-silencing therapies that block the production of endogenous miRNAs.
27.4.6 Aptamers
Aptamers are single- stranded or double- stranded nucleic acids that can bind proteins involved in the
regulation and expression of genes (i.e., transcription factors). In addition, they also bind to proteins that
perform other regulatory functions. For example, a 15- mer (i.e., 15 nucleotide long) DNA aptamer binds
after parenteral administration.
27.4.7 Ribozymes
bond in a single- stranded RNA molecule in a sequence- dependent manner. This process, therefore, leads
to interference with the translation process.
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FIGURE 27.3 Mechanisms of RNA interference. Long double- stranded RNA is cleaved by Dicer into fragments of 21– 23
nucleotide siRNAs. Following unwinding, the antisense strand of duplex siRNA is incorporated into RNA- induced silencing
complex (RISC) protein. Subsequently, the incorporated siRNA stands guides RISC to its homologous target mRNA for
endonucleolytic cleavage.
Pharmaceutical Dosage Forms and Drug Delivery
27.4.8 RNA Interference
ally inhibit gene expression at a posttranscriptional level. Endogenous mRNA exists as a single strand.
chops it up into small fragments of between 21 and 25 base pairs in length. Such a short double- stranded
RNA fragment is called siRNA. The siRNA can bind certain cellular proteins to form the RNA- induced
silencing complex (RISC). The RISC gets activated when the siRNA unwinds. The activated complex
binds to the mRNA corresponding to the antisense RNA. Thus, siRNA silences a target gene by binding
to its complementary mRNA and triggering degradation. The mechanisms of RNAi are illustrated in
Figure 27.3.
peutic strategy. Three ways are commonly used for producing siRNA: chemical synthesis, administration
of plasmid DNA, and viral vectors encoding small hairpin RNA (shRNA) expression cassettes. The transcription of genetic sequence in the plasmid DNA or viral vector leads to the production of an mRNA that
has internal self- complementarity, which leads to the formation of a double- strand with a closed hairpinlike loop at one end (shRNA). The shRNA becomes a substrate for the Dicer, leading to the endogenous
formation of siRNA.
27.5 Gene Therapy
Gene therapy is a method for the treatment or prevention of disease that uses genes to provide the patient’s
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27.6 mRNA- Based Gene Therapy
high- level in- vivo expression and potential for achieving treatment at various levels. mRNA- based
therapeutics use host cell protein translation machinery to express the therapeutic proteins. mRNA
therapeutics offer several advantages. Due to the fact that they do not require entry into the nucleus to
to DNA- based medications. mRNA therapeutics are low toxic and have minimum potential risk of accidental infection or opportunistic insertional mutagenesis. Additionally, mRNA has a wider therapeutic
potential for diseases requiring protein expression than transient traditional protein/ peptide medications.
This is attributed to its continuous translation into encoded proteins/ peptides, which produce longlasting expression. Furthermore, it is possible to generate mRNA in an environment devoid of cells
microorganisms and the quality and safety concerns associated with their production. On top of these,
production compared to conventional drug development approaches. Several mRNA therapies have been
developed for treating cancers, infections, and metabolic disorders in preclinical studies, with some currently in clinical trials. A successful application of the mRNA- based COVID- 19 vaccine demonstrates
the viability of this method.
27.6.1 Components of Therapeutic mRNA
-
ation rate of mRNA.
27.6.1.1 5’ Caps
Eukaryotic mRNA has a cap structure, which is essential for protein synthesis, protective group, and
with varying methylation levels) consists of a 7- methylguanosine connected to the next nucleotide
immune response discrimination against foreign RNA. A cytoplasmic (re)- capping complex has been
27.6.1.2 Poly (A) Tail
In general, poly(A) tails comprise a range of 10– 250 adenine ribonucleotides. The length of poly(A)
correlated with the length of poly(A) tails. A greater quantity of units promotes the development of trans-
27.6.1.3 5’- UTRs and 3’- UTRs
contain regulatory elements involved in pre- mRNA processing, stability, and translation initiation. The
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Pharmaceutical Dosage Forms and Drug Delivery
motifs, collectively called cis- regulatory elements, and RNA- binding proteins or trans- acting factors.
pyrimidine- rich translational element (PRTE), cytosine enriched regulator of translation (CERT) domain,
G- quadruplex structure, and eIF3- binding stem- loop structure.
27.6.1.4 The Open Reading Frame
-
tent in ORFs protects mRNA from endoribonuclease degradation and enhances in vivo protein expression. Functional peptides are essential for mRNA drugs, and signal peptides are necessary for proteins
with external functions.
27.6.2 mRNA Translation Process
Eukaryotic mRNA translation initiation is a controlled process involving a complex of proteins and RNA
that guides ribosomes to the start codon. Poly(A)- binding protein (PABP) attaches to the poly(A) tail and
eIFs are released to start amino acid chain extension.
27.6.3 Three mRNA Posttranslation Decay
Therefore, mRNA degradation involves remodeling the messenger ribonucleoproteins (mRNP) structure,
27.6.4 Self- Amplifying RNA
When it comes to therapies, conventional mRNA has limited action, which necessitates the administration
of several dosages. To overcome this limitation, self- amplifying RNA (saRNA) was designed. The saRNA
molecules derived from alphavirus differ from conventional mRNA in structure. It includes the gene
sequence coding for structural proteins for viruses with the gene sequence of interest. Alphaviruses are
The replicase sequence nsP1- 4 encodes for four different proteins, each with different functions. nsP1
is involved in the process of capping, nsP2 acquires helicase activity, nsP3 is critical for the formation of the replication complex and potentially collaborates with other proteins to impede the inhibitory
mechanisms of the host cell, and nsP4 acquires RNA- dependent RNA polymerase activity. Following
the transfection of saRNA into the cells, translation occurs for each of nsP1- 4 polyproteins, serving as
the precursor for the replicase complex. Following this, nsP2 cleaves nsP1- 4 polyprotein, resulting in the
formation of nsP1- 3 polyprotein and nsP4. The early phase replicase complex transcribes the positivesensed RNA strand into a negative- sensed strand, using the latter as the template for replication. saRNA’s
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other RNA, which reduces the required dosage. However, the main challenge for saRNA is its longer
sequence (usually 9– 12 kb) compared to conventional mRNA.
27.6.5 Circular RNA, Noncoding RNAs, and Competitive Endogenous RNA
Circular RNAs (circRNAs) are another type of single- stranded mRNA molecules that are natural and
form a covalently closed continuous loop. circRNAs have several functions in the cells, such as they
operate as protein traps, scaffolds, and recruiters. They play essential roles in biological processes
by serving as transcriptional regulators, microRNA sponges, and protein templates. circRNA is
a distinctive structure that provides them with increased resistance to RNase degradation and a
prolonged half- life.
Some native and synthetic circRNAs have the ability to encode proteins, which raises the possi-
The absence of a 7- methylguanylate (m7G) end is an intriguing aspect of the translation initiation of
circRNA that distinguishes it from conventional RNA. In contrast, circRNAs start translation via capindependent mechanisms, including internal ribosome entry sites (IRESs). Furthermore, studies show
protein translation from circRNA. A circRNA- based vaccine encoding SARS- CoV- 2 RBD has been
reported recently.
27.6.6 In Vitro Transcription
IVT is a method for converting plasmid DNA templates, or polymerase chain reaction (PCR) templates
into mRNA construct sequences. It involves adding polymerases but requires additional capping dinucleo-
RNA. Two methods are used for capping IVT mRNA: co- transcriptional capping and posttranscriptional
capping. Co- transcriptional capping allows coordinated transcription with mRNA capping, but it has
disadvantages like competitive incorporation of guanosine- 5'- triphosphate (GTP) nucleosides, impairing
while maintaining the mRNA functionality.
27.6.6.1 Synthetic mRNA Purification
to remove immunostimulatory contaminants, free nucleotides, and DNA templates. DNase is used to
and silica membrane column elution are also used, but these methods are unsuitable for scalable or larger
mRNA production. RNase III is used to eliminate dsRNA contaminants and reduce the immunogenicity of
synthetic mRNAs. However, it may cleave the double- stranded secondary structure of mRNA. Cellulose
kb without special equipment. Short and long RNAs can be separated using denaturing polyacrylamide
gel electrophoresis or agarose gel electrophoresis. Strict mRNA quality control standards are crucial for
mRNA therapeutics.
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27.6.6.2 mRNA Therapeutics— Cell Delivery
Pharmaceutical Dosage Forms and Drug Delivery
Synthetic mRNAs are delivered to target cells using lipid delivery vehicles. Lipids serve to safeguard
mRNA molecules against degradation while also promoting their cellular uptake. Lipid nanoparticle
technology advancements are broadening the scope for developing mRNA- based therapeutics. In the
are elaborated.
One important problem for synthetic mRNA is that when it enters cells, it may get entrapped in
to increase its stability and resistance to breakdown. Endosome- disrupting drugs, such as chloroquine or
27.7 Gene Delivery Systems
The promise of gene delivery is to overcome limitations associated with the administration of therapeutic
facturing costs. Two approaches are currently used for gene transfer: viral and nonviral.
27.7.1 Viral Vectors
by a therapeutic gene (called transgene
transduction
to transgene expression. Several different viral vectors have been developed for gene therapy, including
retrovirus, adenovirus, adeno- associated virus (AAV), and herpes simplex virus (HSV). The advantages
and disadvantages of different viral vectors are listed in Table 27.1.
TABLE 27.1
Characteristics of Viral Vectors
Viral Vector
Classication DNA/ RNA
Retrovirus
(MMLV)
Retrovirus
(lentivirus)
Adenovirus dsDNA 7.5 Transient High viral titer, transfects
Adeno- associated
virus (AAV)
Herpes simplex
virus (HSV)
Note: MMLV, Moloney murine leukemia virus.
Insertion
Size (kb) Expression Advantages Disadvantages
RNA 9.0 Stable Integrates, no immune
response
RNA 9.0 Stable Transduces nondividing
cells
both dividing and
nondividing cells
ssDNA 4.5 Stable Little immunogenicity,
integrates
dsDNA 30 Stable Can target neuronal
tissues
Low viral titer, transduces
only dividing cells,
insertional mutations
Low viral titer, transduces
only dividing cells,
insertional mutations
Immunogenic, transient
expression
Low transfection
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AAVs are the most common viral vectors used in the clinic. This is mainly due to their low safety
risk compared to other viral vectors. Adenoviruses and AAVs do not integrate their genes into the host
cell genome. Retroviruses, on the other hand, integrate their genetic material with the host cell genome.
Thus, the duration of gene expression is much longer with retroviruses (weeks to months) as compared to
adenoviruses and AAVs (days to weeks).
27.7.1.1 Retroviral Vector
Retroviral vectors are RNA viruses (i.e., their genome is RNA) possessing the main feature of reverse
transcribing their viral RNA genome into a double- stranded viral DNA. Retroviral vectors can stably
insert into the host DNA. The retroviral genome consists of three encoding regions (portions of DNA
gag region, encoding
polenv region,
region. LTRs are responsible for the regulation and expression of the viral genome.
These vectors can carry foreign genes of <8 kb (kilo base pair length). They carry an inherent risk of
mutagenesis by inserting their genome (called insertion mutagenesis) within a functional gene, which can
compromise the functionality of a critical normal human protein.
Defective retroviral vectors are devoid of the genes encoding viral proteins but retain the ability to
infect cells and insert their genes into the chromosomes of the target cells. Members of this class include
the Moloney murine leukemia viruses (MuLVs) and the lentiviruses.
27.7.1.2 MuLV
MuLV consists of three functional genes: gag, pol, and env
structural genes and inserting therapeutic genes in their place make muLV- based vectors. MuLV- derived
vectors integrate exclusively in dividing cells.
27.7.1.3 Lentiviruses
ability to infect and integrate into nondividing cells has applications for the construction of lentiviral
vectors for gene delivery into nondividing, terminally differentiated cells such as neuronal tissue, hem-
27.7.1.4 Adenoviral Vectors
Adenoviruses are nonenveloped DNA viruses carrying linear double- stranded DNA of about 35 kb in length.
accommodated within the capsid. Adenoviral vectors infect both dividing and nondividing cells. Adenoviral
vectors do not integrate into the host cell chromosomes. Genes introduced into cells using adenoviral vectors
are maintained extrachromosomally in the nucleus and provide transient transgene expression.
E1A). This also creates space for the insertion
of the desired gene. Adenoviral vectors are based on natural adenoviruses of serotypes 2 and 5. In these
E1B and E3 genes can be made to create
more space for transgene insertion.
An advantage of adenoviruses over retroviral vectors is the achievement of very high viral titers. This
the host cell, which permits only transient expression of the therapeutic gene. Furthermore, expression
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27.7.1.5 Adeno- Associated Virus Vectors
Pharmaceutical Dosage Forms and Drug Delivery
AAV is a single- stranded DNA virus and belongs to the family of parvoviruses. For example, AAV- 2 is
a nonpathogenic human virus, and the wild- type AAV- 2 genome establishes a latent infection in human
requires an adenovirus or a herpes virus for viral replication. Compared to adenoviruses, AAV has low
immunogenicity. It has a limited capacity for insertion of foreign genes ranging only from 4.1 to 4.9 kb.
For the construction of rAAV- based vectors, the rep and cap genes (which are responsible for the production of proteins that would replicate the virus or produce structural proteins for the capsid) are replaced
by therapeutic genes.
27.7.1.6 Herpes Simplex Virus Vectors
HSV- 1 is a DNA virus possessing a double- stranded linear genome of 150 kb. The HSV affords a large
packaging capacity for the insertion of foreign genes. HSV- 1 can infect both dividing and nondividing
cells. HSV has a natural tropism toward neuronal cells, and this property can be exploited for gene
therapies for neuronal tumors. HSV- 1 particles are relatively stable and can be concentrated in high
virus titers, which are valuable for the low- volume administration of a large number of viral particles.
The virus does not integrate into the host genome and, therefore, exhibits transient gene expression in
infected cells.
27.7.2 Nonviral Gene Expression System: Plasmid Vectors
erate host immune responses (both cellular and humoral), plasmid- based nonviral vectors are fairly safe.
As illustrated in Figure 27.4, three essential components of gene medicines are a therapeutic gene that
The gene and the gene expression system are the components of plasmid DNA, which is a circular
double- stranded DNA molecule. The basic components of a gene expression plasmid are illustrated in
Figure 27.5. Plasmid- based gene expression systems contain a cDNA sequence coding for a therapeutic
FIGURE 27.4 Basic components of a nonviral gene medicine. Therapeutic genes, gene delivery systems, and gene expression plasmids are the three basic components of a nonviral gene medicine.
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FIGURE 27.5 Basic components of a gene expression plasmid.
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gene and several other genetic elements, including introns, polyadenylation sequences, and transcript
to an expression plasmid, such as a gene switch, which enables the expression of the therapeutic protein
Once inside the cytoplasm, the plasmid can then translocate to the nucleus, where gene expression begins
through the natural cellular processes of transcription and translation.
27.7.3 Nonviral Gene Delivery Systems
Plasmid DNA is a long polyanionic polymer. Depending on the number of base pairs, its hydrodynamic
is a barrier to the cell membrane translocation of the plasmid DNA. This barrier is commonly overcome
most commonly used synthetic gene carriers are cationic polymers and lipids, which condense plasmids
into small particles and protect them from degradation by nucleases. These positively charged lipids,
polymers, or lipopolymers interact with the negatively charged plasmid DNA in aqueous solution to form
condensed colloidal particles with low hydrodynamic diameter and an overall positive charge, which
have higher cellular uptake. The positively charged gene delivery systems can have interactions with
other physiological proteins that are negatively charged, leading to toxicities. The apparent potency of
of the immune system, uptake and adsorption by nontarget cells and structures, access to target tissues,
delivery systems that are safe for repeated administration.
27.7.3.1 Lipid- Based Gene Delivery
Plasmids may be incorporated into cationic or neutral liposomes. With the right selection of lipids for
making liposomes, the liposomes can be made pH sensitive so that they are fusogenic (i.e., fuse with the
cell membrane) at acidic pH. This feature has been used to facilitate the endosomal disruption and subsequent release of plasmids in the cytoplasm. The cellular uptake process involves the incorporation of
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