Transcript Fundamentals of Biotechnology

Fundamentals of Biotechnology
Gene therapy
Principles and applications of
therapy based on
targeted inhibition of gene
expression in vivo
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 One way of treating certain human disorders is to selectively inhibit
the expression of a predetermined gene in vivo.
 In principle, this general approach is particularly suited to treating
cancers and infectious diseases, and some immunological disorders.
 In these cases, the basis of the therapy is to knock out the expression
of a specific gene that allows disease cells to flourish, without
interfering with normal cell function.
 For example, attention could be focused on selectively inhibiting the
expression of a particular viral gene that is necessary for viral
replication, or an inappropriately activated oncogene.
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 In addition to the above, targeted inhibition of gene expression may
offer the possibility of treating certain dominantly inherited
disorders.
 If a dominantly inherited disorder-----of a loss-of-function mutation,
treatment may be difficult using conventional gene augmentation
therapy.
 However, dominantly inherited disorders which arise because of a
gain-of-function mutation may not be amenable to simple addition of
normal genes.
 Instead, it may be possible, in some cases, to inhibit specifically the
expression of the mutant gene, while maintaining expression of the
normal allele.
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 The expression of a selected gene might be inhibited by a variety of
different strategies.
 One possible type of approach involves specific in vivo mutagenesis
of that gene, altering it to a form that is no longer functional.
 Gene targeting by homologous recombination offers the possibility
of site-specific mutagenesis to inactivate a gene.
 Instead, methods of blocking the expression of a gene without
mutating it have been preferred.
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 In principle, this can be accomplished at different levels:
 at the DNA level :
(by blocking transcription);
 at the RNA level:
(by blocking post-transcriptional processing, mRNA transport or
engagement of the mRNA with the ribosomes);
 or at the protein level:
(by blocking post-translational processing, protein export or other steps that
are crucial to the function of the protein).
Targeted inhibition of expression at the DNA level
 Under certain conditions, DNA can form triple-stranded structures, as
occurs naturally in the case of a portion of the mitochondrial genome.
 The rationale of triple helix therapeutics is to design a gene-specific
oligonucleotide that will have a high chance of base-pairing with a defined
double-stranded DNA sequence of a specific target gene in order to inhibit
transcription of that gene.
 Binding of the single-stranded oligonucleotide to a pre-existing double
helix occurs by Hoogsteen hydrogen bonds and certain bases are
preferred.
 The most stable of such bonds are formed by a G binding to the G of a GC
base pair and a T binding to the A of an AT base pair.
Targeted inhibition of expression at the RNA level.
 Antisense
therapeutics involves binding
oligonucleotides or polynucleotides to the RNA;
of
gene-specific
 in some cases, the binding agent may be a specifically engineered
ribozyme, a catalytic RNA molecule that can cleave the RNA
transcript.
Targeted inhibition of expression at the protein level.
 Oligonucleotide aptamers and intracellular antibodies can be
designed to specifically bind to and inactivate a selected
polypeptide/protein .
Antisense oligonucleotides or
polynucleotides can bind to a specific
mRNA, inhibiting its translation and, in
some cases, ensuring its destruction
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 During transcription, only one of the two DNA strands in a DNA
duplex, the template strand (or antisense strand), serves as a
template for making a complementary RNA molecule.
 As a result, the base sequence of the single-stranded RNA transcript
is identical to the other DNA strand (the sense strand), except that
U replacesT.
 Any oligonucleotide or polynucleotide which is complementary in
sequence to an mRNA sequence can therefore be considered to be
an antisense sequence.
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 Binding of an antisense sequence to the corresponding mRNA
sequence would be expected to interfere with translation, and
thereby inhibit polypeptide synthesis.
 Indeed, naturally occurring antisense RNA is known to provide a way
of regulating the expression of genes in some plant and animal cells,
as well as in some microbes.
Antisense oligodeoxynucleotides
 The use of artificial antisense oligodeoxynucleotides is often favored,
simply because:
 they can be synthesized so simply.
 They can be transferred efficiently into the cytoplasm of cells using
liposomes,
 and can migrate rapidly to the nucleus by passive diffusion through
the pores of the nuclear envelope.
 Antisense oligodeoxyribonucleotides (ODNs) are preferred as they
are generally less
oligoribonucleotides.
vulnerable
to
nuclease
attack
than
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 Nevertheless, to protect against degradation by cellular exonucleases
it is still usual to modify the oligonucleotides at their 3′ or 5′ ends
e.g. by introducing more resistant phosphorothioate bonds where
sulfur atoms are linked to phosphate groups instead of the normal
oxygen atoms.
 Antisense ODNs are also preferred because they have the additional
advantage of inducing the destruction of an mRNA to which they
bind.
 This is so because an ODN-mRNA hybrid, like all DNA-RNA
hybrids, is vulnerable to attack by a specific class of intracellular
ribonuclease, RNase H which selectively cleaves the RNA strand.
Peptide nucleic acids
 Peptide nucleic acids (PNAs) are artificially constructed by attaching
the bases found in nucleic acids to a pseudopeptide backbone.
 The normal phosphodiester backbone is entirely replaced with a
polyamide (peptide) backbone composed of 2-aminoethyl glycine
units.
 As a result, PNAs have improved flexibility compared to DNA or
RNA, which permits more stable hybridization to DNA or RNA (by
Watson-Crick hydrogen bonding).
 They are also more resistant to nuclease attack and may therefore be
useful alternatives to conventional antisense oligonucleotides.
Ribozymes
 Some RNA molecules are able to lower the activation energy for
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specific biochemical reactions, and so effectively function as
enzymes (ribozymes).
They contain two essential components:
target recognition sequences (which base-pair with complementary
sequences on target RNA molecules),
and a catalytic component which cleaves the target RNA molecule
while the base-pairing holds it in place.
The cleavage leads to inactivation of the RNA, presumably because
of subsequent recognition by intracellular nucleases of the two
unnatural ends. Examples include human ribonuclease P and various
ribozymes obtained from plant viroids (virus-like particles).
Intracellular antibodies (intrabodies)
 Antibody function is normally conducted extracellularly. Once
synthesized, they are normally secreted into the extracellular fluid,
or are transported to the surface of the B cell to act as an antigen
receptor.
 Recently, however, it has been possible to design genes encoding
intracellular antibodies, or intrabodies.
 Intrabodies can be directed to a particular cell compartment where
they can bind to and inactivate a specific cell molecule such as a
disease-causing protein, and so they have been envisaged to have
potential for treating certain diseases, such as infectious diseases.
Oligonucleotide aptamers
 Oligonucleotide aptamers are oligonucleotides which can bind to
a specific protein sequence of interest.
 Transfer of large amounts of a chemically stabilized aptamer into
cells can result in specific binding to a predermined polypeptide,
thereby blocking its function.
Mutant proteins
 Naturally occurring gain-of-function mutations can involve the
production of a mutant polypeptide that binds to the wild-type
protein, inhibiting its function.
 In many such cases, the wild-type polypeptides naturally associate to
form multimers, and incorporation of a mutant protein inhibits this
process.
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 In some cases, gene therapy may be possible by designing genes to
encode a mutant protein that can specifically bind to and inhibit a
predetermined protein, such as a protein essential for the life-cycle
of a pathogen.
 For example, one form of gene therapy for AIDS involves artificial
production of a mutant HIV-1 protein in an attempt to inhibit
multimerization of the viral core proteins.
Artificial correction of a pathogenic
mutation in vivo is possible, in principle,
but is very inefficient and not readily
amenable to clinical applications
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 Certain disorders are not easy targets for conventional gene therapy.
 For example, dominantly inherited disorders where a simple
mutation results in a pathogenic gain of function cannot be treated
by gene augmentation therapy,
 and targeted inhibition of gene expression may be difficult to
achieve.
 An alternative to conventional gene therapy involves repair of a
mutant sequence in vivo.
 In principle,
this can be done by a variety of different experimental strategies at
both the level of the mutant gene or its transcript.
Therapeutic repair at the DNA level
 One possible approach is to achieve correction of the genetic defect
by therapeutic gene targeting.
 Homologous recombination-based gene targeting low efficiency and
there are formidable challenges in applying this technology to in vivo
gene therapy.
 Other possibilities for therapeutic DNA repair utilize triple helix
formation and peptide nucleic acids.
Therapeutic repair at the RNA level
 An alternative approach to gene targeting is to repair the genetic defect at
the RNA level. One possibility is to use a therapeutic ribozyme.
 One method envisages using a class of ribozyme known as group I
introns,
 which are distinguished by their ability to fold into a very specific shape,
 capable of both cutting and splicing RNA.
 If a transcript has, for example, a nonsense or a missense mutation,
it may be possible to design specific ribozymes that can cut the RNA
upstream of the mutation and then splice in a corrected transcript, a form
of trans-splicing .
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 Another possibility is therapeutic RNA editing.
 This involves using a complementary RNA oligonucleotide to bind
specifically to a mutant transcript at the sequence containing the
pathogenic point mutation,
 and an RNA editing enzyme, such as double-stranded RNA
adenosine deaminase, to direct the desired base modification.
Thank you so much to all of
U