as per PCI Syllabus for M. Pharm (Pharmaceutics) students

1. Nucleic acid basedNucleic acid based
therapeuticstherapeutics
Dr. Atish S. Mundada
Associate Professor,
SNJB’s SSDJ College of Pharmacy,
Chandwad, Dist. Nashik

2. Introduction:Introduction:
• Modern drug research aims to discover biologically active
molecule(s) that are absolutely specific to the molecular targets
responsible for the disease progression.
• Moreover, there is strong belief that medicine will soon benefit
from the development of new therapeutic technologies to directly
target human genes.
• Insertion of new genetic material into the cells of an individual
with the intention of producing a therapeutic benefit for the
patient is human gene therapy.
• Numerous gene therapy strategies are under development, some
of which use nucleic acid-based molecules to inhibit gene
expression at either the transcriptional or posttranscriptional
level.
• A number of human diseases are known to be genetic in origin
and virtually all diseases have a hereditary component and thus,
gene therapy represents an opportunity for the treatment of
these.

3. • Elucidation of the human genome has also provided a major
impetus in identifying human genes implicated in diseases, which
may eventually lead to the development of nucleic acid-based
drugs for gene replacement or potential targets for gene ablation.
• Moreover, the Human Genome project will help determine
genetic markers responsible for patient response to drug therapy,
drug interactions, and potential side effects.
• Currently, all gene therapy trials approved for human use target
somatic cells that will live only as long as the patient, and this is
known as the somatic gene therapy. Its purpose is to alleviate
disease in the treated individual alone.
• In contrast, it is also possible to target directly the gametes (sperm
and ova) to modify the genetic profile of the subsequent
generation of unborn “patients.”
• This gene transfer at an early stage of embryonic development is
known as the germ line gene therapy.

4. • More than 300 clinical trials involving gene transfer in patients
have been approved, and the first nucleic acid drug, an antisense
oligonucleotide, fomivirsen (marketed as Vitravene), has been
approved by the US FDA for the treatment of cytomegalovirus
retinitis in immuno-compromised patients.
• Despite the obvious advantages that might be gained from human
gene therapy (i.e., replacing a defective gene with a normal one),
there are many ethical, social, and commercial issues surrounding
the technology. The outcome of an error in technology might not
be observed for many years.
• Moreover, it is feared that unpredictable and perhaps irreversible
side effects occur in treated individuals.
• The social implications of such technology include the possibility
that patients might suffer from depression as a result of being
“genetically altered” or might not be accepted by society in the
way that they were before treatment.

5. Nucleic acids:Nucleic acids:
• It is a naturally occurring chemical compound that is capable of
being broken down to yield phosphoric acid, sugars and a mixture
of organic bases (purines and pyrimidines).
• Nucleic acids are polymers of acidic monomeric subunits known
as nucleotides.
• Nucleic acids are the main information-carrying molecules of the
cell and by directing the process of protein synthesis, they
determine the inherited characteristics of every living thing.
• The two main classes of nucleic acids are deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA).
• DNA is the master blueprint for life and constitutes the genetic
material in all free-living organisms and most viruses.
• RNA is the genetic material of certain viruses, but it is also found
in all living cells, where it plays an important role in certain
processes such as the making of proteins.
• Nucleic acids represent novel and largely unexploited targets for
therapeutic agents.

6. Nucleotides: building blocks of nucleic acids:Nucleotides: building blocks of nucleic acids:
• DNA and RNA are polymers (in the case of DNA, often very long
polymers), and are made up of monomers known as nucleotides.
When these monomers combine, the resulting chain is called a
polynucleotide (poly- = "many").
• Each nucleotide is made up of three parts:
– a nitrogen-containing ring structure called a nitrogenous base,
– a five-carbon sugar, and
– at least one phosphate group.
• The sugar molecule has a central position in the nucleotide, with
the base attached to one of its carbons and the phosphate group
(or groups) attached to another.
• When the nucleotide joins the growing DNA or RNA chain, it loses
two phosphate groups. So, in a chain of DNA or RNA, each
nucleotide has just one phosphate group.

8. Biosynthesis and Degradation:Biosynthesis and Degradation:
• Nucleotides are synthesized from readily available precursor in the
cell. The ribose phosphate portion of both purine and pyrimidine
nucleotides is synthesized from glucose via the pentose phosphate
pathway.
• The six-atom pyrimidine ring is synthesized first and subsequently
attached to the ribose phosphate. The two rings in purines are
synthesized while attached to the ribose phosphate during the
assembly of adenine or guanine nucleosides.
• Finally, a specialized enzyme called a kinase adds two phosphate
groups using ATP as the phosphate donor to form ribonucleoside
triphosphate, the immediate precursor of RNA.
• For DNA, the 2′-hydroxyl group is removed from the
ribonucleoside diphosphate to give deoxyribonucleoside
diphosphate.
• An additional phosphate group from ATP is then added by another
kinase to form a deoxyribonucleoside triphosphate, the
immediate precursor of DNA.

9. • During normal cell metabolism, RNA is constantly being made and
broken down.
• The purine and pyrimidine residues are reused by several salvage
pathways to make more genetic material.
• Purine is salvaged in the form of the corresponding nucleotide,
whereas pyrimidine is salvaged as the nucleoside.

10. Deoxyribonucleic acid (DNA):Deoxyribonucleic acid (DNA):
• DNA is a polymer of the four nucleotides A, C, G, and T, which are
joined through a backbone of alternating phosphate and
deoxyribose sugar residues.
• These nitrogen-containing bases occur in complementary pairs as
determined by their ability to form hydrogen bonds between
them.
• A always pairs with T through two hydrogen bonds, and G always
pairs with C through three hydrogen bonds. The spans of A:T and
G:C hydrogen-bonded pairs are nearly identical, allowing them to
bridge the sugar-phosphate chains uniformly.
• This structure, along with the molecule’s chemical stability, makes
DNA the ideal genetic material.
• The bonding between complementary bases also provides a
mechanism for the replication of DNA and the transmission of
genetic information.

11. • In 1953 C. Watson and Crick proposed a three-dimensional
structure for DNA based on low-resolution X-ray crystallographic
data.
• They shared a Nobel prize in 1962 for their efforts and postulated
that two strands of polynucleotides coil around each other,
forming a double helix.
• Naturally occurring DNA molecules can be circular or linear. The
genomes of single-celled bacteria (the prokaryotes), as well as the
genomes of mitochondria and chloroplasts are circular molecules.
• In addition, some bacteria and archaea have smaller circular DNA
molecules called plasmids that typically contain only a few genes.
Many plasmids are readily transmitted from one cell to another.
• The genomes of most eukaryotes and some prokaryotes contain
linear DNA molecules called chromosomes.
• Human DNA, for example, consists of 23 pairs of linear
chromosomes containing three billion base pairs.

12. • In all cells, DNA does not exist free in solution but rather as a
protein-coated complex called chromatin.
• In prokaryotes, the loose coat of proteins on the DNA helps to
shield the negative charge of the phosphodiester backbone.
• Chromatin also contains proteins that control gene expression and
determine the characteristic shapes of chromosomes.
• In eukaryotes, a section of DNA between 140 and 200 base pairs
long winds around a discrete set of eight positively charged
proteins called a histone, forming a spherical structure called the
nucleosome.
• Additional histones are wrapped by successive sections of DNA,
forming a series of nucleosomes like beads on a string.
• Transcription and replication of DNA is more complicated in
eukaryotes because the nucleosome complexes have to be at least
partially disassembled for the processes to proceed effectively.

13. DenaturationDenaturation::
• The strands of the DNA double helix are held together by hydrogen
bonding interactions between the complementary base pairs.
• Heating DNA in solution easily breaks these hydrogen bonds,
allowing the two strands to separate-a process called denaturation
or melting.
• The two strands may reassociate when the solution cools,
reforming the starting DNA duplex-a process called renaturation or
hybridization.
• These processes form the basis of many important techniques for
manipulating DNA. For example, a short piece of DNA called an
oligonucleotide can be used to test whether a very long DNA
sequence has the complementary sequence of the oligonucleotide
embedded within it.
• Using hybridization, a single-stranded DNA molecule can capture
complementary sequences from any source. Single strands from
RNA can also reassociate. DNA and RNA single strands can form
hybrid molecules that are even more stable than double-stranded
DNA.

14. Mutations:Mutations:
• Chemical modification of DNA can lead to mutations in the
genetic material.
• Anions such as bisulfite can deaminate cytosine to form uracil,
changing the genetic message by causing C-to-T transitions.
• Exposure to acid causes the loss of purine residues, though
specific enzymes exist in cells to repair these lesions.
• Exposure to UV light can cause adjecent pyrimidines to dimerize,
while oxidative damage from free radicals or strong oxidizing
agents can cause a variety of lesions that are mutagenic if not
repaired.
• Halogens such as chlorine and bromine react directly with uracil,
adenine, and guanine, giving substituted bases that are often
mutagenic.
• Similarly, nitrous acid reacts with primary amine groups-for
example, converting adenosine into inosine-which then leads to
changes in base pairing and mutation.

15. Sequence determination:Sequence determination:
• Methods to determine the sequences of bases in DNA were
pioneered in the 1970s by F. Sanger and W. Gilbert, whose efforts
won them a Nobel Prize in 1980.
• The Gilbert-Maxam method relies on the different chemical
reactivities of the bases, while the Sanger method is based on
enzymatic synthesis of DNA in vitro.
• Both methods measure the distance from a fixed point on DNA to
each occurrence of a particular base—A, C, G, or T.
• DNA fragments obtained from a series of reactions are separated
according to length in four “lanes” by gel electrophoresis. Each
lane corresponds to a unique base, and the sequence is read
directly from the gel.
• The Sanger method has now been automated using fluorescent
dyes to label the DNA and a single machine can produce tens of
thousands of DNA base sequences in a single run.

16. • RNA, unlike DNA, is usually single-stranded.
• A nucleotide in an RNA chain will contain ribose (the five-carbon
sugar), one of the four nitrogenous bases (A, U, G, or C), and a
phosphate group.
• RNA is made by copying the base sequence of a section of double-
stranded DNA, called a gene, into a piece of single-stranded
nucleic acid. This process, called transcription, is catalyzed by an
enzyme called RNA polymerase.
• RNA is a relatively unstable nucleic acid that serves a variety of
functions in cells including providing structure to higher order
complexes, enzymatic activity and as a template for protein
synthesis.
• There are four major types of RNA: messenger RNA (mRNA),
ribosomal RNA (rRNA), transfer RNA (tRNA), and regulatory RNAs.
• The rRNA represents approximately 80% of cellular RNA, tRNA
approximately 15%, and mRNA 3–5%.
Ribonucleic acid (RNA):Ribonucleic acid (RNA):

17. • In prokaryotes the protein coding sequence occupies one
continuous linear segment of DNA. However, in eukaryotic genes
the coding sequences are frequently “split” in the genome—a
discovery reached independently in the 1970s by R. Robert and
Philip Sharp, whose work won them a Nobel Prize in 1993.
• The segments of DNA or RNA coding for protein are called exons,
and the non-coding regions separating the exons are called
introns.
• Following transcription, these coding sequences must be joined
together before the mRNAs can function.
• The process of removal of the introns and subsequent rejoining of
the exons is called RNA splicing.
• Each intron is removed in a separate series of reactions by a
complicated piece of enzymatic machinery called a spliceosome.

18. Types of RNA:Types of RNA:
1. Messenger RNA (mRNA)- is an intermediate between a protein-
coding gene and its protein product. If a cell needs to make a
particular protein, the gene encoding the protein will be turned
“on,” meaning an RNA-polymerizing enzyme will come and make
an RNA copy, or transcript, of the gene’s DNA sequence.
• However, in the RNA molecule, the base T is replaced with U.
• Once an mRNA has been produced, it will associate with a
ribosome, a molecular machine that specializes in assembling
proteins out of amino acids.
• The ribosome uses the information in the mRNA to make a protein
of a specific sequence, “reading out” the mRNA’s nucleotides in
groups of three (called codons) and adding a particular amino acid
for each codon.
• In general, prokaryotic mRNAs are degraded very rapidly, whereas
the cap structure and the polyA tail of eukaryotic mRNAs greatly
enhance their stability.

19. 2. Ribosomal RNA (rRNA)- molecules are the structural components
of the ribosome.
• The rRNAs form extensive secondary structures and play an active
role in recognizing conserved portions of mRNAs and tRNAs. They
also helps mRNA bind in the right spot so its sequence information
can be read out.
• Some rRNAs also act as enzymes, meaning that they help
accelerate (catalyze) chemical reactions – in this case, the
formation of bonds that link amino acids to form a protein.
• RNAs that act as enzymes are known as ribozymes.
• In the prokaryote E. coli, seven copies of the rRNA genes synthesize
about 15,000 ribosomes per cell.
• In eukaryotes anywhere from 50 to 5,000 sets of rRNA genes and
as many as 10 million ribosomes may be present in a single cell.
• In eukaryotes these rRNA genes are looped out of the main
chromosomal fibres and coalesce in the presence of proteins to
form an organelle called the nucleolus.

20. 3. Transfer RNAs (tRNAs)- carries individual amino acids into the
ribosome for assembly into the growing polypeptide chain.
• Their job is to act as carriers – to bring amino acids to the
ribosome, ensuring that the amino acid added to the chain is the
one specified by the mRNA.
• The tRNA molecules contain 70 to 80 nucleotides and fold into a
characteristic cloverleaf structure.
• Specialized tRNAs exist for each of the 20 amino acids needed for
protein synthesis, and in many cases more than one tRNA for each
amino acid is present.
• The amino acids are loaded onto the tRNAs by specialized
enzymes called aminoacyl tRNA synthetases, usually with one
synthetase for each amino acid.
• All tRNAs adopt similar structures because they all have to interact
with the same sites on the ribosome.

21. 4. Regulatory RNA (miRNAs and siRNAs)- Some types of non-coding
RNAs (RNAs that do not encode proteins) help regulate the
expression of other genes. Such RNAs may be called regulatory
RNAs.
• For example, microRNAs (miRNAs) and small interfering RNAs
siRNAs are small regulatory RNA molecules about 22 nucleotides
long.
• They bind to specific mRNA molecules (with partly or fully
complementary sequences) and reduce their stability or interfere
with their translation, providing a way for the cell to decrease or
fine-tune levels of these mRNAs.
• These are just some examples out of many types of noncoding and
regulatory RNAs.
• Scientists are still discovering new varieties of noncoding RNA.

22. RibozymesRibozymes::
• Not all catalysis within the cell is carried out exclusively by
proteins. Thomas Cech and Sidney Altman, jointly awarded a Nobel
Prize in 1989, discovered that certain RNAs, now known as
ribozymes, showed enzymatic activity.
• They showed that the RNA component of an RNA protein complex
called ribonuclease P can cleave a precursor tRNA to generate a
mature tRNA. In addition to self-splicing RNAs, artificial RNAs have
been made that show a variety of catalytic reactions.
• It is now widely held that there was a stage during evolution when
only RNA catalyzed and stored genetic information.
• This period, sometimes called “the RNA world,” is believed to have
preceded the function of DNA as genetic material.

23. Antisense RNAs:Antisense RNAs:
• Most antisense RNAs are synthetically modified derivatives of RNA
or DNA with potential therapeutic value.
• In nature, antisense RNAs contain sequences that are the
complement of the normal coding sequences found in mRNAs (also
called sense RNAs).
• Like mRNAs, antisense RNAs are single-stranded, but they cannot
be translated into protein.
• They can inactivate their complementary mRNA by forming a
double-stranded structure that blocks the translation of the base
sequence.
• Artificially introducing antisense RNAs into cells selectively
inactivates genes by interfering with normal RNA metabolism.

24. Other RNAs:Other RNAs:
• Many other small RNA molecules with specialized functions are
present in cells.
• For example, small nuclear RNAs (snRNAs) are involved in RNA
splicing and other small RNAs that form part of the enzymes
telomerase or ribonuclease P are part of ribonucleoprotein
particles.
• Other RNA molecules serve as guide RNAs for editing, or they are
complementary to small sections of rRNA and either direct the
positions at which methyl groups need to be added or mark U
residues for conversion to the isomer pseudouridine.

25. Nucleic acid metabolism:Nucleic acid metabolism:
DNA metabolism-
• Replication, repair, and recombination—the three main processes
of DNA metabolism—are carried out by specialized machinery
within the cell.
• DNA must be replicated accurately in order to ensure the integrity
of the genetic code.
• Errors that creep in during replication or because of damage after
replication must be repaired.
• Finally, recombination between genomes is an important
mechanism to provide variation within a species and to assist the
repair of damaged DNA.

26. RNA metabolism-
• RNA provides the link between the genetic information encoded
in DNA and the actual workings of the cell.
• Three distinct phases of RNA metabolism occur.
• First, selected segments of the genome are copied by transcription
to produce the precursor RNAs.
• Second, these precursors are processed to become functionally
mature RNAs ready for use. When these RNAs are mRNAs, they
are then used for translation.
• Third, after use the RNAs are degraded, and the bases are
recycled.
• Thus, transcription is the process where a specific segment of
DNA, a gene, is copied into a specific RNA that encodes a single
protein or plays a structural or catalytic role.
• Translation is the decoding of the information within mRNA
molecules that takes place on a specialized structure called a
ribosome.

27. • One of the crowning achievements of molecular biology was the
elucidation during the 1960s of the genetic code.
• Principals in this effort were Har G Khorana and Marshall W
Nirenberg , who shared a Nobel Prize in 1968.
• Khorana and Nirenberg used artificial templates and protein
synthesizing systems in the test tube to determine the coding
potential of all 64 possible triplet codons.
• The key feature of the genetic code is that the 20 amino acids are
encoded by 61 codons. Thus, there is degeneracy in the code such
that one amino acid is often specified by more than one codon.
• The structure within the genetic code whereby many amino acids
are uniquely coded by the first two bases of the codon strongly
suggests that the code has itself evolved from a more primitive
code involving 16 dinucleotides.

28. NucleicNucleic--acid based gene therapeutics:acid based gene therapeutics:
• The ability to alter or transform cellular physiology via the delivery
of exogenous nucleic acid molecules to cells has been a common
research tool in the laboratory for decades to study gene
functions.
• By the 1980s, the concept of gene therapeutics has moved from
the bench side to the bedside, when a series of clinical trials
demonstrated therapeutic efficacy from the transplantation of
virally transduced cells.
• The power of gene therapy is derived from the ability to
manipulate cell physiology at genetic and epigenetic levels,
accessing molecular processes that are previously unreachable by
conventional pharmacological means.
• This allows particular pathways and factors to be targeted with
unparalleled specificity, thereby greatly improving the efficacy in
therapy and dramatically reducing side effects commonly
associated with wide spectrum pharmacological compound.

29. • As is common to all drug development processes, delivery is the
foremost challenge for gene therapeutics.
• The large molecular weight and anionic charges prohibits nucleic
acid molecules from entering the cell via passive diffusion across
the negatively charged lipid bilayer of the plasma membrane, and
thus calls for a facilitated uptake process.
• This challenge was initially met by engineering disarmed
retroviruses, whose virulence factors that enable viral replication
have been removed from the viral genome and replaced with the
nucleic acid sequences coding for a protein with therapeutic
potential.
• The development of synthetic nonviral gene delivery systems has
been met with various technical and biological challenges.
• Several types of cationic polymers and lipids have been explored
for this purpose with varying levels of transfection efficiencies.

30. • Nucleic acids can be modified through genetic recombination to
insert functional elements that can self-modulate its own activities
ranging from target specificity, bioavailability, intracellular
trafficking, to regulated expression and sustained protein
production, all without affecting the integrity or competency of the
nucleic acid.
• Unlike conventional pharmacological compounds, nucleic acids are
delivered as a prodrug, where the activity, instructed by nucleic
acid sequences, would depend on the physiology of the cell
carrying out those instructions.
• An inherent disconnect between the pharmacokinetics of the
nucleic acid complexes and the kinetics of the expressed transgene
product is natural.
• Determining the correlation between delivery efficiencies and
therapeutic efficacy would necessary involve retooling of existing
methods.

31. Types of genetic modifications:Types of genetic modifications:
• Gene-based therapy is most commonly associated with gene
augmentation or gene replacement therapy, where the deficient
gene product is supplied with a functional version.
• Our expanding knowledge of molecular genetics has broadened
avenues of gene therapy to include gene inhibition, editing and
repair.
• While the mode of genetic modifications differs in their effects on
gene of interest, the biological outcome is largely dependent on
the identity of the targeted gene.
• These modifications are not mutually exclusive in their application
and can work in parallel or in concert to achieve the same
therapeutic outcome.
• Three ways through which genetic modification can be achieved-
– Gene augmentation
– Gene knockdown
– Gene repair

32. Gene augmentation:
• In gene augmentation therapy (GAT), the aim of the treatment is
to enhance the amount of protein product by delivering
exogenous nucleic acid molecules containing instruction for the
expression of the deficient protein.
• This type of therapy is typically applied to correct monogenic loss-
of-function mutations that underlay many metabolic and
physiological disorders, which are conventionally treated with
protein-based enzyme or hormone supplement.
• Beyond inherited or chronic metabolic disorders, GAT has been
applied to cancer therapy via de novo expression of suicide genes
that have the ability to induce cell death following its own
expression and can range from apoptosis inducer, such as
caspases to enzymes that converts prodrugs to cytotoxic
compound.
• In addition, the augmented expression of tumor antigen epitope
in cancer cells has been demonstrated to enhance its
immunogenicity and anti-tumor activity of cytotoxic T cell.

33. • The clinical utility of GAT is diverse and is highly dependent on the
activity of the expressed protein, but in all cases, the objective is
to get transgene production from the expression construct.
• The most common nucleic acid for GAT is bacterialderived,
mammalian expression plasmid DNA (pDNA).
• These DNA molecules typically range in sizes from 3 to 10
kilobases and contain coding sequences for the gene of interest,
promoters, enhancers, and polyadenylation sites, which are
crucial to the expression and posttranscriptional processing of the
transgene mRNA.
• Beyond these mammalian elements, the remainder of the
molecules is occupied by bacterial sequences required for
replication, partition and selection of the pDNA during clonal
expansion of the molecule from a bacterial host.
• pDNA is a popular choice for GAT owing to well-established
methods that allow convenient insertion and removal of
sequences to modularize the performance and activity of the
vector with genetic elements.

34. • An alternative approach to bypassing the need for nuclear entry is
through the delivery of mRNA instead of pDNA, which can be
directly translated in the cytoplasm.
• A significant drawback of this approach is that RNA molecules are
highly susceptible to degradation by extracellular and intracellular
nucleases.
• However, recent advances in both in vitro transcription and
chemical modification have significantly improved the stability and
synthesis scale of RNA that is feasible for gene therapy.
• Beyond pDNA, development of artificial chromosomes (AC) has
gained significant momentum toward clinical readiness.
• The major advantage of AC is their ability to be replicated and
maintained autonomously as an episome, which allows transgene
expression to be sustained in subsequent generations.
• The capacity for the size of the transgene in AC is also much
greater than any vector system currently available.

35. Gene knockdown:
• Gene knockdown refers to the downregulation of gene expression
at either the transcriptional or translational levels.
• This is typically applied to reverse the deleterious effects caused by
the abnormal expression of a mutated protein, an oncogene or a
virulence factor.
• Gene knockdown in mammalian cells can be mediated through a
number of natural processes.
• RNA interference (RNAi) is a posttranscriptional silencing pathway
that uses short stretches of double-stranded RNA (dsRNA)
molecules as inducers.
• The dsRNA are cleaved by a Rnase III-like protein called the “Dicer
protein”, to yield shorter 21–23 nt molecules with 2-nucleotide 3′-
overhang at both ends, known as small interfering RNA (siRNA);
siRNA are then bound to RNA-induced silencing complexes (RISC)
where the sense strand is cleaved, allowing the antisense strand to
guide the complex through a homologydependent base pairing
through a degradation pathway, thereby preventing the translation
of the mRNA into protein

36. • RNAi can be induced by exogenously delivered dsRNA (siRNA),
whose activity is located in the cytosol. The silencing activity,
however, is transient as degradation consumes the siRNA.
• Alternatively, pDNA containing either the RNA Pol II or Pol III
promoter have been used to drive the transcription of small
hairpin RNA (shRNA), an intermediate in the RNA processing
pathway and a precursor of siRNA, for more sustained silencing
activity.
• However, nuclear import is required for the transcription of shRNA
and may therefore limit its silencing efficiency.
• The siRNA molecules can be considered as a substrate for the
enzymatic processes in gene silencing.
• As with protein engineering, chemical constituents on the
oligonucleotides can be “re-engineered” to alter this lock-and-key
binding interaction. Indeed, a growing repertoires of chemical
modifications have been applied to siRNA molecules in an effort to
improve their efficacy, potency, serum stability, specificity, and
delivery as well as modulating their immunogenicity and
minimizing off-target effect.

37. • Gene knockdown can further be mediated through an RNAi-
independent mechanism involving antisense oligonucleotides (As-
ODN).
• As-ODN are 18–21 nt single stranded DNA molecules that share
complementary sequences to its target gene.
• Hybridization of the As-ODN to target sequences inhibits gene
expression via several mechanisms.
• Similar to siRNA, As-ODNs are chemically modified to enhance
their efficacy and stability while warding off attacks from
nucleases.
• The new generation of chemical moieties and stereoisomers
improve upon the previous generation by further enhancing
binding affinity, target specificity and nuclease resistance.

38. Gene repair-
• Gene replacement is the ideal corrective approach, achieving
stable and accurate genome integration remains a technical
challenge at present; the risk of ectopic integration often
outweighs the therapeutic benefits.
• For this reason, there is great interest in gene repair as a method
for restoring wild type functions in dominant negative mutations.
• Repair can be implemented at the mRNA level or at the genome
level by editing out miscoded sequences through nucleotide base
transition, splice site modulation, mismatch repair using antisense
oligomers, or oligonucleotide-mediated genome editing.
• RNA editing via base transition is based on the premise that
nucleotide can be covalently modified to change its
complementary base pairing properties. For example, the
deamination of adenosine and cytosine result in inosine and uracil,
respectively.

39. • RNA editing is initiated by binding of antisense oligomer to the
mutated target mRNA to form double-stranded structures.
• The RNA duplex structures are recognized by adenosine
deaminase acting on RNA (ADARs) and cytosine deaminase acting
on RNA (CDARs) to catalyze the base modifications.
• RNA editing is a naturally occurring event utilized by cells for
posttranscriptional processing . While RNA editing is limited to A-
to-G and C-to-U base transition, ADAR can act on not only pre-
mRNA, but viral RNA and noncoding miRNA as well. Thus, it has
therapeutic potential in wide range of diseases.
• In contrast, gene editing at the genome level provides a long-term
solution to gene correction and is based on the observation that
DNA fragments with homologous sequences to a target locus can
induce site-specific recombination or single-base mismatch repair.
• Various types of DNA molecules ranging from triplex-forming
oligonucleotides, RNA/DNA hybrid oligonucleotides and small
DNA fragments have been applied to genome editing in model
diseases.

40. Barriers to nucleic acid based therapeutics:Barriers to nucleic acid based therapeutics:
• The simplicity in using nucleic acid based therapeutics comes
from the fact that structurally and chemically identical nucleic
acid molecules can impart and modulate a wide array of
activities-As-ODN can be used for gene editing, RNA repair and
gene knockdown; pDNA can be adapted to transcribe protein-
encoding mRNA, or transcript-modulating shRNA.
• The complexity and challenge then arise in accurately identifying
the molecular targets, specifying their activities through
sequences and delivering the molecules to appropriate
subcellular compartments in which the activity is to be carried
out.
• The list of barriers will likely differ for each type of carrier,
delivery platform (ex vivo vs. in vivo), the types of nucleic acid
cargo (DNA vs. RNA) and the types of genetic modification
intended (expression vs. inhibition vs. repair).

41. DNADNA--Based Therapeutics:Based Therapeutics:
• Plasmids:- Plasmids are high molecular weight, double-stranded
DNA constructs containing transgenes, which encode specific
proteins.
• On the molecular level, plasmid DNA molecules can be considered
prodrugs that upon cellular internalization employ the DNA
transcription and translation apparatus in the cell to biosynthesize
the therapeutic entity, the protein.
• The mechanism of action of plasmid DNA requires that the plasmid
molecules gain access into the nucleus after entering the
cytoplasm.
• Nuclear access or lack thereof eventually controls the efficiency of
gene expression.
• In addition to disease treatment, plasmids can be used as DNA
vaccines for genetic immunization.

42. • Oligonucleotides for Antisense and Antigene Applications:-
Oligonucleotides are short single-stranded segments of DNA that
upon cellular internalization can selectively inhibit the expression
of a single protein.
• For antisense applications, oligonucleotides interact and form a
duplex with the mRNA or the pre-mRNA and inhibit its translation
or processing, consequently inhibiting protein biosynthesis.
• For antigene applications, oligonucleotides must enter the cell
nucleus, form a triplex with the double-stranded genomic DNA,
and inhibit the translation as well as the transcription process of
the protein.
• For therapeutic purposes, oligonucleotides can be used to
selectively block the expression of proteins that are implicated in
diseases.
• Antisense oligonucleotides such as MG98 and ISIS 5132, designed
to inhibit the biosynthesis of DNA methyltransferase and c-raf
kinase respectively, are in human clinical trials for cancer.

43. • Aptamers:- DNA aptamers are double-stranded nucleic acid
segments that can directly interact with proteins.
• Aptamers interfere with the molecular functions of disease-
implicated proteins or those that participate in the transcription or
translation processes.
• Aptamers are preferred over antibodies in protein inhibition owing
to their specificity, nonimmunogenicity, and stability of
pharmaceutical formulation.
• DNA aptamers have demonstrated promise in intervention of
pathogenic protein biosynthesis against HIV-1 integrase enzyme.

44. • DNAzymes:- DNAzymes are analogs of ribozymes with greater
biological stability.
• The RNA backbone chemistry is replaced by the DNA motifs that
confer improved biological stability.
• DNAzyme directed against vascular endothelial Nucleic Acids as
Therapeutics 23 growth factor receptor 2 was confirmed to be
capable of tumor suppression by blocking angiogenesis upon
intratumoral injections in mice.

45. RNARNA--Based Therapeutics:Based Therapeutics:
• RNA Aptamers:- RNA aptamers are single-stranded nucleic acid
segments that can directly interact with proteins.
• Aptamers recognize their targets on the basis of shape
complementarity.
• Moreover, their binding specificity and affinity for the target are
extremely high and similar to monoclonal antibodies.
• RNA aptamers have demonstrated promise in intervention of
pathogenic protein biosynthesis against HIV-1 transcriptase.
• Moreover, RNA aptamers that specifically bind and inactivate
vascular endothelial growth factor (VEGF) in vitro have been
isolated.
• A clinical study on humans with injection of anti-VEGF aptamers in
the eye showed that 80% of the patients retained or improved the
eyesight, and they had no side effects.

46. • RNA Decoys:- The RNA decoys are designed to provide alternate,
competing binding sites for proteins that act as translational
activators or mRNA-stabilizing elements.
• Decoys can prevent translation or induce instability and,
ultimately, destruction of the mRNA.
• Overexpressed short RNA molecules corresponding to critical cis-
acting regulatory elements can be used as decoys for trans-
activating proteins, thus preventing binding of these trans
activators to their corresponding cis-acting elements in the viral
genome.
• RNA decoys have an advantage over other nucleic acid-based
strategies, that is, the decoys are less likely to be affected by
variability of the infectious agent because any mutation in the
trans-activating protein affects not only binding to the decoys but
also binding to the endogenous targets.

47. • Antisense RNA:- Antisense drugs are short stretches of
deoxyribonucleotide analogs that bind to specific complementary
areas of the mRNA by Watson–Crick base pairing to block gene
expression in a sequence-specific fashion.
• Antisense drugs may induce an RNaseH, which cleaves the mRNA
at the site of binding, or can physically block translation or other
steps in mRNA processing and transport to protein synthesis.
• The antisense drugs work at an early stage in the production of a
disease-causing protein and theoretically can be applied to a
number of diseases where the basic pathophysiology involves an
overexpression of a given protein molecule.
• A stoichiometric disadvantage of antisense RNA is the high
expression required to successfully bind to all target RNA.
• A major advantage is the lack of immunogenicity of antisense
constructs, such that the oligonucleotides and the cells producing
them will not be destroyed by the host immune response.

48. • Ribozymes:- Antisense RNA alone is not potent enough to
produce complete inhibition in vivo.
• An enzymatic moiety can be included with antisense
oligonucleotide, which will cleave the target RNA once the RNA–
RNA duplex has formed.
• These enzymatic RNA strands are called “Ribozymes.” They are
antisense RNA molecules that are capable of sequence-specific
cleaving of RNA molecules.
• They function by binding to the target moiety through antisense
sequence-specific hybridization and inactivating it by cleaving the
phosphodiester backbone at a specific site.
• Thus, they can selectively bind to target mRNAs and form a duplex
having highly distorted confirmation that is easily hydrolyzed, and
this hydrolysis of mRNA may be used for targeted suppression of
specific gene.

49. • Two types of ribozymes, the hammerhead and hairpin ribozymes
(the names are derived from their theoretical secondary
structures), have been extensively studied owing to their small size
and rapid kinetics and for therapeutic applications.
• Hammerhead ribozymes cleave RNA at the nucleotide sequence
U–H (H ¼ A, C, or U) by hydrolysis of a 30–50 phosphodiester bond,
while hairpin ribozymes utilize the nucleotide sequence C–U–G as
their cleavage site.
• The presence of the RNA backbone in ribozymes makes them easy
targets for degradation by RNases, so these molecules are
biologically unstable in vivo.
• Ribozymes can be used for knockout gene therapy by targeting
over expressed oncogenes such as the human epidermal growth
factor receptor type-2 gene implicated in breast cancer and human
papilloma virus infection.
• A significant limitation of the use of ribozymes for gene therapy is
that they are susceptible to RNases.

50. • Small Interfering RNAs:- RNA interference (RNAi) is a
posttranscriptional mechanism of gene silencing through
chromatin remodeling, inhibition of protein translation, or direct
mRNA degradation, which is ubiquitous in eukaryotic cells.
• Small interfering RNAs (siRNAs) can be used for downregulation of
disease-causing genes through RNA interference.
• Typically, these are short double-stranded RNA segments with 21–
23 nucleotides and are complementary to the mRNA sequence of
the protein whose transcription is to be blocked.
• On administration, siRNA molecules are incorporated into RNA-
induced silencing complexes (RISC), which bind to the mRNA of
interest and stimulate mRNA degradation mechanisms, such as
nuclease activity, that lead to silencing of the particular gene.
• Introduction of foreign double-stranded RNAs (dsRNA) can initiate
a potent cascade of sequence-specific degradation of endogenous
mRNAs that bear homology to the dsRNA trigger.

51. • MicroRNA:- miRNAs are a class of naturally occurring, small
noncoding RNA molecules 21–25 nucleotides in length.
• These molecules are partially complementary to messenger RNA
(mRNA) molecules, and their main function is down regulation of
gene expression via translational repression, mRNA cleavage and
deadenylation.