*3.2.2 General delivery methods*

OD delivery can occur through carrier molecules. Receptor-mediated endocytosisdirected uptake utilizes import mechanisms already present in the cell membrane for the uptake of biomolecules necessary for cell function [67]. ODs can be linked directly to a carrier protein *via* a covalent bond or non-covalently via poly-L-lysine (PLL)-carrier conjugates. The choice of the carrier is dependent on its known ability to bind to specific cell membrane receptors and accumulate in the cell via endocytosis. Not only OD's internalization can be potentially improved, but cell-specific delivery can also be achieved by targeting receptors exclusively expressed or over-expressed on certain target cells. However, the OD is generally sequestrated in endosomal compartments, thereby limiting the utility of this method for delivery [22]. The use of peptides can help overcome this issue. Indeed, as peptides have membrane translocation properties, they can increase ODs passage through the plasma membrane. For example, fusogenic peptides have been used to promote peptide fusion of OD-peptide conjugates with either cell or lysosomal membranes [68]. To facilitate OD transport to the nucleus, nuclear localization signals (NLS) can be used for ODs that inhibit pre mRNA splicing [69]. Another peptide used is CPP, a short peptide sequence

**23**

toxicity in animal models.

*Antisense Oligonucleotides, A Novel Developing Targeting Therapy*

(<30 amino acids) with net positive charge that uses an energy dependent pathway to allow internalization of large molecules like ASOs. Commonly used CPP include penetratin, HIV TAT peptide and transportan. Through disulfide bond, ASOs and CPP can be conjugated. To evaluate ASO-CPP effect on mRNA degradation *in vitro* and *in vivo,* PNA is the most used [43, 70, 71]. An interesting variation is to couple two different ASOs to a single CPP thus addressing two targets simultaneously [72]. The most dramatic advance in OD targeting has involved delivery *via* the asialoglycoprotein receptors (ASGR). ASGPR, also known as the Ashwell receptor, is a lectin that is abundantly expressed on hepatocytes [73] and clears serum glycoproteins by receptor-mediated endocytosis [74]. The functional receptor is a trimer comprised of two proteins and exhibits high affinity for *N*-acetyl galactosamine (GalNAc) terminated oligosaccharides [75]. A major breakthrough came from researchers who developed multivalent GalNac conjugated siRNAs that bind to the ASGR [76]. The conjugates were effectively taken up into primary mouse hepatocytes by a receptorspecific mechanism, leading to silencing of targeted genes. GalNac based conjugates have similarly been used to transport ASOs to the liver in mice with good effects on reduction of target gene expression [77]. Indeed, GalNac-ASOs showed high affinity for mouse ASGPR, which resulted in enhanced ASO delivery to hepatocytes. Furthermore, once inside the cell, GalNac-ASO's metabolism leads to ASO release in the liver, thus acting like a prodrug targeting hepatocytes. Moreover it improved potency and extent the effect of both antisense targeting human transthyretin (TTR) and human apolipoprotein C-III in transgenic mice. This study highlighted several of the virtues of conjugates including use of a molecularly defined entity, high tissue and cell selectivity and lack of substantial toxicity. The successes with glycoconjugates in the laboratory have facilitated their rapid translation to clinical evaluation, with several hepatic genes being addressed. Finally, an additional approach to OD internalization is to generate transient permeabilization of the plasma membrane and allow naked ODs to penetrate into the cells by diffusion. It consists of inducing transitory pores in the membrane, either chemically by streptolysin-O-permeabilization, mechanically by microinjection, or by electroporation [78] or ultrasound.

All of these methods, under defined circumstances, can permit charged or uncharged ODs to enter cells rapidly and localize in the nucleus, where they pro-

Nevertheless, one of the most promising strategies used to overcome cellular barriers is the use of nanotechnology. Indeed, nanocarriers offer the possibility to encapsulate drugs but also nucleic acid, to protect it from degradation, enhance its distribution to the tissues and the cells, but also reduce toxicity and thus secondary effects by targeting specific organs. It is a very promising tool for combination therapy and theranostics applications. To bypass the limitations of ASO-based therapy, like insufficient stability and low intracellular delivery and distribution, Dr. Rocchi's lab recently developed a modified first-generation ASO by using a lipid-conjugated oligonucleotide modification (LASO) able to overcome the problems to ASO administration. The idea is to improve ASO's stability, efficiency and biodisponibility by adding a hydrophobic lipid chain at the 5'end of the hydrophilic DNA sequence. As amphiphiles, LASOs self-assemble in aqueous media with an encapsulation rate of 100%, which yields in small spherical objects of ~11 nm in diameter [79]. Due to their small size, these nanomicelles, can accumulate inside the tumor *via* EPR effect (enhanced permeability and retention effect), which permits passive targeting and reduced systemic toxicity. Interestingly, transfection with these lipid-modified ASO leads to rapid and prolonged internalization *via* micropinocytosis with no transfecting agent. Interestingly, the addition of the lipid, does not affect LASO's efficacy *in vitro* and *in vivo* with little or no

*DOI: http://dx.doi.org/10.5772/intechopen.82105*

duce antisense inhibition of gene function.

#### **Figure 3.**

*Cellular uptake and intracellular trafficking of oligonucleotides. Oligonucleotides enter cells via several endocytotic pathways. Most of ODs are recycled in early recycling endosomes. ODs accumulate in endosomes, multivesicular bodies (MVB) and lysosomes. A significant amount is compartmentalized in cellular organelles like the trans Golgi network (TGN).*

#### *Antisense Oligonucleotides, A Novel Developing Targeting Therapy DOI: http://dx.doi.org/10.5772/intechopen.82105*

*Antisense Therapy*

concentrations of ODs.

*3.2.2 General delivery methods*

site of action. In cultured cells, the internalization of naked ASOs is generally inefficient, with only a few ASO molecules actually penetrating the cell [21]. Exogenously administered ASOs enter cells *in vitro* by a combination of fluid-phase (pinocytosis), caveolae potocytosis, adsorptive and receptor-mediated endocytosis (**Figure 3**), cellular uptake mechanisms being different depending on the ASO chemical structure. However, this results in a trafficking problem because not all of the internalized ASO will be able to reach their target and interact with it. This is because the majority of internalized ASO is sequestered into endosome or lysosomal compartments [21]. A significant amount of the OD is also compartmentalized within other cellular organelles, such as the Golgi complex and the endoplasmic reticulum [64].

To improve cellular uptake and OD's activity, a range of techniques and transporters have been developed [65, 66]. Simultaneously, the use of these vectors increases the stability of ODs against nuclease digestion and allows the use of lower

OD delivery can occur through carrier molecules. Receptor-mediated endocytosisdirected uptake utilizes import mechanisms already present in the cell membrane for the uptake of biomolecules necessary for cell function [67]. ODs can be linked directly to a carrier protein *via* a covalent bond or non-covalently via poly-L-lysine (PLL)-carrier conjugates. The choice of the carrier is dependent on its known ability to bind to specific cell membrane receptors and accumulate in the cell via endocytosis. Not only OD's internalization can be potentially improved, but cell-specific delivery can also be achieved by targeting receptors exclusively expressed or over-expressed on certain target cells. However, the OD is generally sequestrated in endosomal compartments, thereby limiting the utility of this method for delivery [22]. The use of peptides can help overcome this issue. Indeed, as peptides have membrane translocation properties, they can increase ODs passage through the plasma membrane. For example, fusogenic peptides have been used to promote peptide fusion of OD-peptide conjugates with either cell or lysosomal membranes [68]. To facilitate OD transport to the nucleus, nuclear localization signals (NLS) can be used for ODs that inhibit pre mRNA splicing [69]. Another peptide used is CPP, a short peptide sequence

*Cellular uptake and intracellular trafficking of oligonucleotides. Oligonucleotides enter cells via several endocytotic pathways. Most of ODs are recycled in early recycling endosomes. ODs accumulate in endosomes, multivesicular bodies (MVB) and lysosomes. A significant amount is compartmentalized in cellular organelles* 

**22**

**Figure 3.**

*like the trans Golgi network (TGN).*

(<30 amino acids) with net positive charge that uses an energy dependent pathway to allow internalization of large molecules like ASOs. Commonly used CPP include penetratin, HIV TAT peptide and transportan. Through disulfide bond, ASOs and CPP can be conjugated. To evaluate ASO-CPP effect on mRNA degradation *in vitro* and *in vivo,* PNA is the most used [43, 70, 71]. An interesting variation is to couple two different ASOs to a single CPP thus addressing two targets simultaneously [72]. The most dramatic advance in OD targeting has involved delivery *via* the asialoglycoprotein receptors (ASGR). ASGPR, also known as the Ashwell receptor, is a lectin that is abundantly expressed on hepatocytes [73] and clears serum glycoproteins by receptor-mediated endocytosis [74]. The functional receptor is a trimer comprised of two proteins and exhibits high affinity for *N*-acetyl galactosamine (GalNAc) terminated oligosaccharides [75]. A major breakthrough came from researchers who developed multivalent GalNac conjugated siRNAs that bind to the ASGR [76]. The conjugates were effectively taken up into primary mouse hepatocytes by a receptorspecific mechanism, leading to silencing of targeted genes. GalNac based conjugates have similarly been used to transport ASOs to the liver in mice with good effects on reduction of target gene expression [77]. Indeed, GalNac-ASOs showed high affinity for mouse ASGPR, which resulted in enhanced ASO delivery to hepatocytes. Furthermore, once inside the cell, GalNac-ASO's metabolism leads to ASO release in the liver, thus acting like a prodrug targeting hepatocytes. Moreover it improved potency and extent the effect of both antisense targeting human transthyretin (TTR) and human apolipoprotein C-III in transgenic mice. This study highlighted several of the virtues of conjugates including use of a molecularly defined entity, high tissue and cell selectivity and lack of substantial toxicity. The successes with glycoconjugates in the laboratory have facilitated their rapid translation to clinical evaluation, with several hepatic genes being addressed. Finally, an additional approach to OD internalization is to generate transient permeabilization of the plasma membrane and allow naked ODs to penetrate into the cells by diffusion. It consists of inducing transitory pores in the membrane, either chemically by streptolysin-O-permeabilization, mechanically by microinjection, or by electroporation [78] or ultrasound.

All of these methods, under defined circumstances, can permit charged or uncharged ODs to enter cells rapidly and localize in the nucleus, where they produce antisense inhibition of gene function.

Nevertheless, one of the most promising strategies used to overcome cellular barriers is the use of nanotechnology. Indeed, nanocarriers offer the possibility to encapsulate drugs but also nucleic acid, to protect it from degradation, enhance its distribution to the tissues and the cells, but also reduce toxicity and thus secondary effects by targeting specific organs. It is a very promising tool for combination therapy and theranostics applications. To bypass the limitations of ASO-based therapy, like insufficient stability and low intracellular delivery and distribution, Dr. Rocchi's lab recently developed a modified first-generation ASO by using a lipid-conjugated oligonucleotide modification (LASO) able to overcome the problems to ASO administration. The idea is to improve ASO's stability, efficiency and biodisponibility by adding a hydrophobic lipid chain at the 5'end of the hydrophilic DNA sequence. As amphiphiles, LASOs self-assemble in aqueous media with an encapsulation rate of 100%, which yields in small spherical objects of ~11 nm in diameter [79]. Due to their small size, these nanomicelles, can accumulate inside the tumor *via* EPR effect (enhanced permeability and retention effect), which permits passive targeting and reduced systemic toxicity. Interestingly, transfection with these lipid-modified ASO leads to rapid and prolonged internalization *via* micropinocytosis with no transfecting agent. Interestingly, the addition of the lipid, does not affect LASO's efficacy *in vitro* and *in vivo* with little or no toxicity in animal models.
