The Intrathecal delivery of compounds acting on central nervous system (CNS) has enhanced interest due to the absence of continuous barriers between the cerebrospinal fluid (CSF) and CNS, which makes only fraction of CSF borne compounds delivered to the brain from the CSF through the perivascular channels. This is significant for macromolecules including biopharmaceuticals, for which the access to CNS from the systemic circulation is particularly hindered due to large molecular size and presence of several vascular barriers such as blood brain, blood-arachnoid and blood-CSF. In the clinical practice, the most routinely practiced routes for accessing the CSF are Intrathecal (IT) lumbar route and more invasive intracerebroventricular (ICV) route through brain parenchyma. Dosing through Intrathecal is less invasive and chronically used with one dose injections directly intra lumbar region for several months over ICV route.
The intrathecal route is currently of significant interest for testing the efficacy of novel compounds in pre-clinical models as well as for clinical use.
The increasing interest in antisense therapies was based on the introduction of the exogenous fragment of nucleic acid, complementary to the mRNA may be an efficient inhibitor of the translation process. Such DNA or RNA fragments are called “antisense oligonucleotides” (ASOs) since they bind via Watson–Crick base pairing to the sense strand of target RNA. ASOs are synthetic, single-stranded compounds, typically built of several dozen nucleotides. The ASO structure should be chemically modified since phosphodiester oligonucleotides are rapidly digested by intracellular enzymes such as endo- and exonucleases. Moreover, native oligonucleotides have a very small affinity to proteins present in the blood (e.g., albumin), which resulted in their fast elimination from the bloodstream. To increase their stability, enhance tissue distribution, and binding affinity to the target sequences, modifications are introduced into sugar moieties, bases or phosphodiester linkages. Oligonucleotides with modified phosphodiester linkages belong to the first generation of ASOs. Such modification involves the replacement of one of the non-bridging oxygen atoms by other atom or chemical group such as e.g., methyl one. Phosphorothioate oligonucleotides (PS) with the oxygen substituted by sulfur atom are the most commonly investigated ASOs. The second generation of these compounds includes modification within sugar moieties. In this case, the hydroxyl group ribose is replaced with a fluorine atom or methyl and methoxyethyl groups (ME and MOE), which significantly reduce polarity. Third generation ASOs usually contains different modification in phosphate groups, sugar moieties as well as in nucleobases. N30 / N50 phosphoramidates, peptide nucleic acids (PNA), morpholino phosphoroamidates (PMOs) as well as locked nucleic acid (LNA) are examples of this generation of ASOs. The study of ASOs biotransformation is especially important since some of their metabolism products may be toxic. For this reason, the evaluation of nonclinical toxicology with the use of animal models is essential to understand the undesirable effects of potential antisense drugs.
The antisense therapy concept is based on the probability that all RNA or DNA sequences longer than 13 and 17 nucleotides occur only once in the human genome. ASOs may be designed to bind not only to the RNA but also DNA, proteins, or other molecules. Based on the mechanism of action, the oligonucleotide therapeutics may be divided into ASOs, siRNA, miRNA as well as aptamers. However, the most popular mechanism of their action includes gene silencing with the use of ASOs. Inhibition of translation may be achieved in various ways, including RNA degradation by RNase H. Moreover, another approach include splicing inhibition and translational arrest. Gene silencing may also be induced by the activity of small RNA fragments including siRNA and miRNA. These RNA fragments bind with RNA-induced silencing complex (RISC), naturally occurring in cells that possess enzymatic activity due to the presence of Ago2 protein. An active RISC complex with an incorporated single strand of siRNA or miRNA can recognize target mRNA, which is then destroyed by Ago2.A most important ASOs mechanism of action is based on RNase H enzyme activity. It is present mainly in the nucleus, however, it can exist also in cytoplasm and mitochondria. This endonuclease can destroy the RNA strand in mRNA/ASO duplex by hydrolytic mechanism. Therefore, released ASO is then able to bind with another copy of mRNA. Thus, the number of targeted RNA is reduced, which consequently leads to a decrease in the level of the target protein. It should be noted that modification type significantly influences the mechanism of ASOs activity and only some medications are able to activate enzymatic cleavage mediated by RNase H. One of the ASOs which promotes RNase H cleavage of target sequences are PS ASO. Such modification not only increases ASOs' resistance against nucleases, allowing them to reach target RNA sequence but also increases their stability in tissue and plasma. Moreover, they are able to destabilize heteroduplex with RNA.
However, PS ASOs have some limitations, regarding specificity, cellular uptake toxicology, and binding affinity to the target sequences. Another approach of the downregulation of mRNA expression by ASOs, is the translational arrest of the targeted protein. ASOs are designed to bind with the translation initiation codon of mRNA and inhibit protein translation. It should be noted that in this case, chemical modification of ASOs also influences the mechanism of action. Most ASOs which are capable to create a steric block are RNase H independent. This group of modification includes changes in the furanose ring structure, such as 20-O-methyl and 20-O-methoxyethyl ASOs, and LNAs, PNAs, and morpholino. They increase ASO stability and cellular uptake as well as the binding affinity to the target sequences.
The lumbosacral area will be shaved and cleaned aseptically. Animal will be anaesthetized by inhalation of isoflurane/air mixture (5% for induction, 2% for maintenance) given at 300 ml/min flow rate using a small animal anesthesia system. The rat will then be positioned on a heating pad at 37°C in sternal recumbency with both pelvic limbs brought as far forward as possible to arch the distal vertebral spine. A disposable 26G hypodermic needle filled with test solution will inserted at 90° to the spine between the L4 and L5 and then tilted at 45° and injected. For successful IT location of the needle will be confirmed by the presence of at least one of the following signs: twitch of the tail and/or presence of cerebrospinal fluid (CSF) in the needle hub. If none of these signs will be seen, or if blood will be visible in the needle hub, the needle will be withdrawn, and the same procedure will be repeated with another needle. If the needle is correctly located, test item (volume: 0.1mL per rat) will be injected at approximately 1 ml/min.
Optimizing the administration dose and improving the therapeutic efficacy of ASOs must be considered to maximize clinical efficacy. Choosing the optimal delivery route is be delivered into the entire neuraxis. The intrathecal route does not require brain surgery. Therefore, serious complications involving brain surgery such as needle tract injury, infection, and hemorrhage can be avoided. Another, the medical cost and psychological burden associated with surgical procedures will be reduced. Finally, the intrathecal route can be beneficial when applied to neurogenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia combined with motor neuron disease, because the route covers not only the brain but also the spinal cord.
CSF will be collected by piercing the cisterna magna with 26 G needle percutaneously. Animals will be anesthetized by inhalation of isoflurane/air mixture (5% for induction) given at 300 ml/min flow rate using small animal anaesthesia platform. The back of the neck and the base of the skull will be shaved and disinfected with 70% ethyl alcohol. The rat neck will be flexed downward at approximately 135°. The location of the cisterna magna is identified by palpation of a 3 mm 2 rhomboid depressed area (atlanto – occipital joint) in the middle of the line connecting the ear bases. The 26G needle will be inserted to depth of 5 -7 mm into the cisterna magna for CSF collection. The needle will be immediately removed after the appearance of the clear CSF.