Project on Morpholino

1. Background:
By 1984 a growing concern in the then-emerging antisense field was that antisense therapeutics might never be commercially viable because of their very high production costs.
Two key factors in the high cost of DNA analogs are:
1) The limited availability and high cost of their deoxyribonucleoside precursors;
2) The complexity and expense associated with coupling to hydroxyls, required in forming the phosphoester intersubunit linkages of most nucleic acid analogs.

In 1985 Summerton devised the Morpholino structural type to circumvent both of these cost problems. This is achieved by starting with much less expensive ribonucleosides and introducing an amine.
1. Introduction:
In molecular biology, a Morpholino is a molecule used to modify gene expression.
Morpholino oligonucleotides (oligos) are an antisense technology used to block access of other molecules to specific sequences within nucleic acid. Morpholinos block small (~25 base) regions of the base-pairing surfaces of ribonucleic acid.

Morpholinos are usually used as a research tool for reverse genetics by knocking down gene function. This is achieved by preventing cells from making a targeted protein or by modifying the splicing of pre-mRNA. Knocking down gene expression is a powerful method for learning about the function of a particular protein; similarly, causing a specific exon to be spliced out of a protein can help to determine the function of the protein moiety encoded by that exon. These molecules have been applied to studies in several model organisms, including mice, zebrafish, frogs, and sea urchins.
Morpholinos are also in development as pharmaceutical therapeutics targeted against pathogenic organisms such as bacteria or viruses and for amelioration of genetic diseases. These synthetic oligos were conceived by James E. Summerton (Gene Tools, LLC) and developed in collaboration with Dwight D. Weller ( AVI BioPharma Inc. )
2. Structure:
Morpholinos are synthetic molecules which are the product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA by standard nucleic acid base-pairing. Structurally, the difference between Morpholinos and DNA is that while Morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through phosphorodiamidate groups instead of phosphates.
Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.
Morpholinos are most commonly used as single-stranded oligos, though heteroduplexes of a Morpholino strand and a complementary DNA strand may be used in combination with cationic cytosolic delivery reagents.







4. Why Choose Morpholinos?
There is no other gene knockdown reagent (including siRNA, PNA, mPNA, S-DNA, and LNA) that combines the properties of stability, nuclease-resistance, efficacy, long-term activity, water-solubility, low toxicity and exquisite specificity. Only Morpholino oligos provide all of these.
Morpholinos provide much better specificity than RNAi, siRNA and phosphorothioate based oligos, greatly decreasing the chance of catastrophic off-target antisense effects.
The exquisite specificity of Morpholinos contributes to their extremely low toxicity and suitability for use in embryos, while off-target gene modulation makes siRNA unusable in most embryonic systems.
The non-ionic backbone of a Morpholino minimizes interactions with proteins, eliminating this mechanism for inducing non-antisense effects. Morpholinos do not trigger cellular responses by binding to Toll-like receptors, eliminating a confounding artifact associated with knockdowns using siRNA.
As steric blocking oligos, Morpholinos can be used not only to block translation but also to alter mRNA splicing, to bind miRNAs or to block binding of miRNA or regulatory proteins to RNA targets. Splice-blocking Morpholinos allow researchers to delete targeted exons and even analyze specific splice-forms of a gene with multiple splice variants. Morpholinos can target a pri-miRNA or a pre-miRNA to inhibit miRNA maturation or target a miRNA or one of its targets to inhibit miRNA activity. Binding of a splice-regulatory protein can be prevented by protecting its binding site with a Morpholino oligo.
5. Properties:
i. Solubility
An antisense oligo should have good water solubility to assure good access to its target sequence within the cell. Conventional perception in the antisense field is that non-ionic antisense oligos (such as Morpholinos) invariably show poor water solubility. Contrary to this perception, it has been found that if the nucleobases in a non-ionic antisense oligo stack poorly then that oligo has poor water solubility, but if the nucleobases are well stacked in aqueous solution then that oligo can show excellent water solubility.
It is assumed that the low water solubility of non-ionic oligos with poorly stacked bases is a result of the difficulty of inserting the hydrophobic faces of the unstacked bases into an aqueous environment. On the other hand, the high water solubility of non-ionic phosphorodiamidate-linked Morpholinos is likely due to effective shielding of those hydrophobic faces from the polar solvent because of the exceptionally good stacking of the bases.

ii. Stability
For optimal activity, an antisense oligo should be completely resistant to nucleases. DNA antisense oligos are degraded in serum and within cells in a matter of minutes.
Morpholino oligos are completely resistant to nucleases, as well as being resistant to a broad range of other degradative factors in biological systems - even including a liver homogenate. Accordingly, Morpholinos are effective in even long-term experiments and they are free of complications, which could arise from toxic degradation products.
iii. Efficiency: Cell-Free
It has been found that in a cell-free translation system with added RNase H the "old" 25-mer Morpholinos containing uracils typically exhibited slightly higher efficacies than corresponding S-DNAs, while "new" thymine-containing 25-mer Morpholinos generally achieve substantially higher efficacies than corresponding S-DNAs.
iv. Efficiency: In Cells
In experiments with Morpholinos carried out at ANTIVIRALS Inc. it has been found that Morpholino antisense oligos, which exhibit good activity in a cell-free translation system also, exhibit correspondingly good activity when scrape-loaded into cultured animal cells.
v. Binding Affinity
Targeting Morpholino oligos is vastly simpler than targeting DNA-based antisense oligos. Morpholinos usually work on the first attempt if they are successfully delivered to the cytosol. DNA and S-DNA oligos have a low probability of shutting down a target gene on the first attempt; it is common for five or six oligos to be tried before an effective sequence is found.
This is in part because a Morpholino oligo has a higher binding affinity than an equivalent DNA-based antisense oligo. This higher affinity allows Morpholinos to invade RNA secondary structure and substantially increases the probability of designing effective oligos. Precisely targeting a single gene is also simplified because Morpholino oligos act via steric-blocking as opposed to an RNAse H-mediated mRNA degradation mechanism.



6. Function of Morpholinos
Morpholinos do not degrade their target RNA molecules, unlike many antisens structural types (e.g. siRNA). Instead, Morpholinos act by "steric blocking", binding to a target sequence within an RNA and simply getting in the way of molecules that might otherwise interact with the RNA.

Normal gene expression in eukaryotes

Eukaryotic gene expression without intervention by a Morpholinos





In eukaryotic organisms, pre-mRNA is transcribed in the nucleus, introns are spliced out, then the mature mRNA is exported from the nucleus to the cytoplasm. The small subunit of the ribosome usually starts by binding to one end of the mRNA and is joined there by various other eukaryotic initiation factors, forming the initiation complex. The initiation complex scans along the mRNA strand until it reaches a start codon, and then the large subunit of the ribosome attaches to the small subunit and translation of a protein begins. This entire process is referred to as gene expression; it is the process by which the information in a gene, encoded as a sequence of bases in DNA, is converted into the structure of a protein. A morpholino can modify splicing or block translation, depending on the morpholino's base sequence.




i. Blocking translation
Translation blocked by a Morpholino oligo

Bound to the 5'-untranslated region of messenger RNA (mRNA), morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein.Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism.

ii. Modifying pre-mRNA splicing
Splicing blocked by a Morpholino oligo

Morpholinos can interfere with pre-mRNA processing steps by:
• preventing splice-directing small nuclear ribonucleoproteins (snRNP) complexes from binding to their targets at the borders of introns on a strand of pre-mRNA
• by blocking the nucleophilic adenine base and preventing it from forming the splice lariat structure
• by interfering with the binding of splice regulatory proteins such as splice silencer and splice enhancers.
Splice modification can be conveniently assayed by reverse-transcriptase polymerase chain reaction.
iii. Blocking other mRNA sites
Morpholinos have been used to block miRNA activity and maturation.They can also block ribozyme activity. Morpholinos targeted to "slippery" mRNA sequences within protein coding regions can induce translational frameshifts.Activities of morpholinos against this variety of targets suggest that morpholinos can be used as a general-purpose tool for blocking interactions of proteins or nucleic acids with mRNA.


7. Applications of Morpholinos:
There are three main applications of morpholinos which are:

a) Blocking mRNA translation
b) Blocking nuclear processing
c) Anti-sense therapeutics
Let us explain them one by one.

a) Blocking translation:
Bound to the 5'-untranslated region of messenger RNA (mRNA), Morpholinos can interfere with progression of the ribosomal initiation complex from the 5' cap to the start codon. This prevents translation of the coding region of the targeted transcript (called "knocking down" gene expression). This is useful experimentally when an investigator wishes to know the function of a particular protein.Morpholinos provide a convenient means of knocking down expression of the protein and learning how that knockdown changes the cells or organism. Some Morpholinos knock down expression so effectively that, after degradation of preexisting proteins, the targeted proteins become undetectable by Western blot.

b) Blocking nuclear processing:
Morpholinooligos can block nuclear processing events, pre-mRNA processing in particular. The power of high specificity and steric blocking allows one to specifically and reproducibly delete exons of choice by blocking access of the splicing machinery to the pre-mRNA. This technology, not possible with RNase-dependent or RISCdependentoligos (phosphorothioates, RNAi and others), not only allows characterizing specific exon function and creating loss-of-function deletions or insertions but it also allows researchers to eliminate a specific splice variant while leaving anothersplice variant of the same gene intact.

c) Anti-sense therapeutics:
Highly complementary antisense morpholino oligonucleotides (AMOs) can bind to pre-mRNA and modulate splicing site selection. This offers a powerful tool to regulate the splicing process,
such as correcting subtypes of splicing mutations and nonsense mutations and reprogramming alternative splicing processes. Therefore, AMO-mediated splicing modulation represents an attractive therapeutic strategy for genetic disorders. Primary immunodeficiency diseases (PIDs) are a heterogeneous group of genetic disorders that result from mutations in genes involved in development and maintenance of the immune system. Many of these mutations are splicing mutations and nonsense mutations that can be manipulated by AMOs.






8. DELIVERY OF MORPHOLINOS

To make a morpholino effective we have to effectively deliver it to the cytosol through the cell membrane. In cytosol they can freely diffuse and start functioning. Different methods are used for delivering morpholinos, some of which are mentioned below:

i. Delivery to Embryo:

Microinjection:
A microinjection apparatus is usually used for delivery into an embryo, with injections most commonly performed at the single-cell or few-cell stage.

Electroporation:
An alternative method for embryonic delivery is electroporation, which can deliver oligos into tissues of later embryonic stages.

“Electroporation is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field. It is usually used in molecular biology as a way of introducing some substance into a cell, such as loading it with a molecular probe, a drug that can change the cell's function, or a piece of coding DNA.”



ii. Delivery to Cultured cells:

One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems.
So as a solution a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electro statically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol.

iii. Delivery to Adult cells:

Use of cell-penetrating peptides to help Morpholinos into cells, primarily through an endocytotic pathway followed by release from the endosome.

Systemic delivery into many cells in adult organisms can be accomplished by using covalent conjugates of Morpholino oligos with cell-penetrating peptides, and, while toxicity has been associated with moderate doses of the peptide conjugates, they have been used in vivo for effective oligo delivery at doses below those causing observed toxicity. An octa-guanidinium dendrimer attached to the end of a Morpholino can deliver the modified oligo (called a Vivo-Morpholino) from the blood to the cytosol. Delivery-enabled Morpholinos, such as peptide conjugates and Vivo-Morpholinos, show promise as therapeutics for viral and genetic diseases.


9. Vivo morpholinos


Vivo-Morpholinos are the knockdown, exon-skipping or miRNA blocking reagent of choice for in vivo experiments. Outstanding results can be achieved systemically with intravenous injection, and modest systemic delivery achieved with Vivo-Morpholinos by intraperitoneal (I.P.) injection. Efficient localized delivery can be achieved by injecting the Vivo-Morpholinos directly into the area of interest. If you wish to initially validate the oligo in cultured cells you can use the same Vivo-Morpholinos in cultures. However, unmodified Morpholinos, delivered with either electroporation or Endo-Porter, are more efficient in cell cultures than Vivo-Morpholinos



i. Composition:

A Morpholino molecule consists of a Morpholino oligo with a unique covalently linked delivery moiety, which is comprised of an octa-guanidine dendrimer. It uses the active component of arginine rich delivery peptides (the guanidinium group) with improved stability, low toxicity and reduced cost.
The Vivo-Morpholino is assembled by coupling the vivo-delivery group to a Morpholino while the oligo is still bound to its synthesis resin, allowing excellent purification by washing the solid-phase resin. Vivo-Morpholinos must be chosen prior to synthesis and cannot be added later because the vivo-delivery group is added to a Morpholino prior to cleavage from its synthesis resin.

ii. Delivery and dosage:

For best systemic delivery results the injection method of choice is I.V. although I.P. can also be used. The maximum suggested dosage in mammals is 12.5 mg/kg in a 24 hour period. This can be repeated daily. The dosage for short term and long term experiment differ. For short term the experiment lasts 2-3 days the dosage can be 12.5 mg/kg. While for the long term experiments the dosage is preferably started at dosage equivalent to the short term. The dosage and duration can be increased to optimize the results.
One can expect quantifiable knockdown or exon-skipping in liver, small intestine, colon, muscle, lung, and stomach tissues with lesser but quantifiable delivery in the spleen, heart, skin and brain.
It has been found that younger or older mice do not tolerate Vivo-Morpholinos as well and may require substantially lower doses. In addition the use of compromised mice, such as those with less robust genetic backgrounds, may require further dose limitations.

10. Problem Assosiated with Morpholinos:

Occasionally Morpholino oligos can fail and there are many factors which can contribute to oligo failure, some correctable ones determined.

1) Morpholino Oligos can lose activity if improperly stored or stored in anything other than sterile pure water. It needs to be stay fluffy and smooth, and should not turn hard palette like. To avoid this, if the oligo is to be stored long term keep it in a desiccator at room temperature. If the oligo was fluffy and there is still problems resuspending it, it may be due to high G content or reduced solubility due to an added moiety such as lissamine fluorescent tag. In these cases, try heating to 65C for 10 minutes and vortexing. Leaving the oligo overnight on a vigorous shaker might also help dissolution and Continue until completely resuspended.

2) Stock Morpholino oligo should be dissolved in sterile water without Diethylpyrocarbonate (DEPC) because DEPC forms bond with morphiline ring in the oligos backbone. It is possible for an oligo to lose activity if they have undergone long-term exposure to acid or DEPC.

3) Some highly expressed transcripts, like actin, may be difficult to shut down irrespective of oligo concentration because of high expressivity.

4) Specificity is reduced as concentration is increased. For this reason it is important to deliver only enough oligo to achieve near-quantitative shutdown without affecting non-specific targets.

5) There are other factors that can contribute to reduced activity which include potential secondary oligo targets and secondary structure within an oligo. The secondary targets can come from homologous genes. Secondary structure of an oligo can have a significant impact on activity. However, the concern is much greater for inter-strand pairing between oligos than intra-strand pairing within an oligo since single-molecule stem-loops do not readily form due to the oligo's limited flexibility, but inter-strand pairing can tie-up oligo and thus eliminate it from the pool of oligo that can pair with target sequence.

6) We cannot target point mutations with morpholinos. The property which gives Morpholino oligos their spectacular sequence specificity also largely precludes their use for effectively targeting point mutations. However, Morpholinos are the best tools for studying point mutations, polymorphisms, and a host of other genetic variations using the gene switch strategy or splice-blocking.

7) we cannot target the amino acid coding region of mRNA with morpholinos, because Morpholino oligos functions solely by a steric blocking mechanism, only the leader sequence and the first 25 bases of the amino acid coding region can be targeted for translation blocking applications.

8) Morpholino oligos enter unperturbed tissue culture cells but high concentrations (tens of µM) and long incubation times (multiple days) are required to achieve even very modest amounts of delivery in most cell types.

9) The following cells can NOT be delivered with the Morpholinos
a. All T-cell derived lines (apparently do not endocytose)
b. All differentiated colon carcinoma cell lines (also apparently do not endocytose)

10) Non-ionic Morpholinos will not complex with cationic lipids for delivery
11. Alternates of morpholino:


i. Small interfering RNA (siRNA):
Also known as short interfering RNA or silencing RNA, is a class of double-stranded RNA molecules, 20-25 nucleotides in length, that play a variety of roles in biology. The most notable role of siRNA is its involvement in the RNA interference (RNAi) pathway, where it interferes with the expression of a specific gene. siRNAs were first discovered by David Baulcombe's group at the Sainsbury Laboratory in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants. The group published their findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants".







ii. MicroRNAs (miRNAs):
MicroRNAs are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long and are found in all eukaryotic cells, except fungi, algae, and marine plants. miRNAs are post-transcriptional regulators that bind to complementary sequences on target messenger (mRNAs), usually resulting in translational repression and silencing. The human genome may encode over 1000 miRNAs, which may target about 60% of mammalian gene and are abundant in many human cell types.




Reference:

http://www.gene-tools.com/node/12

http://network.nature.com/groups/drugdelivery/forum/topics/359

http://nar.oxfordjournals.org/content/early/2007/08/01/nar.gkm478.full.pdf+html

http://www.reference.com/browse/morpholino

http://morpholinos.yuku.com/

http://www.gene-tools.com/node/25

http://www.gene-tools.com/faq