Diseases of the CNS remain the world’s leading cause of disabilities, accounting for more hospitalization and prolonged care than almost all other diseases combined. Scuh CNS diseases include brain tumours, HIV encephaopathy, epielpsy, verebrovascular diseases and neurodegnerative disorders. The incidence of CNS disorders in human increases with age. Being the most delicate organ of the body, the brain is protected against potentially toxic substances by the blood-brain barrier (BBB); however, the BBB also prevents the penetraiton of most moleules that have potential activity in the CNS. 98% of small molecules are not able to cross the BBB and 100% of large molecuels cannot cross the BBB. (Amin “Getting into the brain” CNS Drugs, 2009, 35-58),
The highly restrictive nature of the BBB results in the majority of small polar molecules being incapable of dif- fusing into the brain (Figure 3C). These molecules include glucose, amino acids, nucleosides and many other mol- ecules. Given the essential roles these molecules play in cell growth and metabolism, it is essential for them to be transported across the BBB. This is achieved through the presence of carrier mediated transport proteins pre- sent on both the luminal and abluminal membranes of the endothelial cells of the BBB. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
Present at all levels of the vascular tree, the BBB is com- posed of endothelial cells lining the brain vasculature cou-pled with surrounding astrocytes and pericytes. Forming the morphological basis of the BBB, brain endothelial cells are phenotypically dissimilar to those of the peripheral circulation (Bernacki et al, 2008). Brain endothelial cells are characterised by their highly polarised tight junctions connecting them to adjacent cells. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
The highly polarised nature of the BBB endothelial cells and the overall tightness of the structure, restricts the trans- port of molecules greater than 500 daltons into the brain (Daneman, 2012). This results in the prevention of an over- whelming majority of small molecules from crossing the BBB (Gabathuler, 2010). In addition to this, the few drugs capable of crossing the barriers are actively pumped back out by protein efflux transporters present on the endothe- lial cells membranes. (Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
Given the significant need to increase cytotoxic therapy accumulation in metastatic brain tumours, there are numerous strategies to enhance delivery which could be investigated. These approaches include hijacking recep- tor mediated transport mechanisms present on the BBB endothelium, bypassing the BBB through local delivery of therapeutic agents, the delivery ochemotherapeutics simultaneously with drug transport inhibitors and disruption of the barrier. Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
Viral Vector Delivery:
To circumvent the blood brain barrier, most genomic medicines rely on direct intracranial injection of viral vectors encoding the transgene of interest. Viral vectors, such as recombinant adeno-assocaited virus (AAv) have had great success in gene therapy and are less immunogenic than most viral vectors; however, they require re-manufacturing for each target and are hindered by costly producton scale-up. In addition, AAv has a limited DNA packaging capacity and is assocaited with immunogenicity in the brain from both the vector and expression of foreign transgenes. Although the brain has been considered an immune privileged site, green fluorescent prtoein can induce a strong inflammatory response and neuronal cell death 3 weeks after injection with AAV serotype 9. In addition, Cas9 specific immune response have been elicited following AAV delviery in mice and pre-existing cellualr and humoral immunity ot Cas9 and AAVs are documented in humans. (Stah. “Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs” Molecular Therapy).
Receptor-Mediated Transport:
Receptor-mediated transport (RMT) is how large endogenous molecules, such as neuropeptides (insulin, transferrin or leptin) corrs the BBB. Insulin uptake is for example mediated by the insulin receptor, transferrin uptake is mediated by the transferrin receptor (Tfr), insulin-like growth factor (IGF) upstake is medaited by the IGF recptor and leptin uptake is medated by the leptin receptor. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),
Designing genetically engineered fusion proteins to make use of the RMT to transport molecuels across the BBB is one approach for brain targeting. These proteins are prepared by fusing human transferrin to mouse-human chimeric IgG3 at 3 positons. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),
The human insulin receptor (HIR), the most potent molecular trjan hourse in the BBB, is active in humans and rehesus monkeys. Brain drug delivery in the rhesus monkey was acheived by attaching a murine 83-14 mAb to the HIR. Amin “Getting into the brain” CNS Drugs, 2009, 35-58),
Fusion constructs:
Stah. (“Genome editing in the mouse brain with minimally immunogenic Cas9 RNPs” Molecular Therapy) disclsoes enablement of self-devliery of the Cas9 ribo nuclear prtoeins. Four repeats of the positively charged Simian vacuolating virus 40 nuclear localizaiton sequences (SV40-NLS) were fused to the N temrinus along with two repeats to the C terminus of Cas9. Using a single guid to turn on the tdTomato reported form the lox-stlop-lox Ai9 mouse, they reproted edited striatal volcume of about 1.5 nm.
Antibody-Mediated Transport:
Given the strong expression of the TfR on the BBB, numer-ous therapeutics have been generated in an attempt to improve the therapeutic treatment of Alzheimer’s disease and glioblastoma (Xu et al, 2011; Yu et al, 2011). In 2011, Xu et al reported the anti-tumour effects of an anti-Tf mono-clonal antibody on glioma cells in vitro, both in combination with a chemotherapeutic drug and alone (Xu et al, 2011). Interestingly, results from this study showed that the anti- body alone had an anti-proliferative effect through induced S phase accumulation and apoptosis, and when used in combination with the chemotherapeutic the effect was fur- ther enhanced, which suggested that combination therapy was more effective. Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
Olson (WO 2018/119001) discloses peptide-antibody fusions/conjugates for targeting therapetuic agents in the CNS or across the blood brain barrier. Olson teaches that the peptide or the antibody of the complex homes or targets to the crebrospinal fluid or hippcampus.
Sengupta (US 2017/0014528) discloses targetd drug delivery conjugates that include a targeting moiety linked ot a drug via a molecule haivng affinity for the targeting moeity (affinity ligand). Typically the conjugate includes a targeting ligand and a molecule of interest such as a therapetuic agent. The tareting ligand and molecule of itnerest are linked to each other via an affintiy ligand. The affintiy ligand is further covalently or non-covalently linked to a drug or therapetuic agent.
Antibodies targetting Transferrin receptor (Tfr):
Antibodies targeting the TfR which is highly expressed by endothelial cells that mkae up the BBB have been reported to cross the BBB, but whether endocytosis of anti-TfR antibodies into the brain vasculature can lead to antibody release and accumulation in the brain parenchyma remains controversial. (Dennis, “Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target” 2011).
Dennis, (“Boosting brain uptake of a therapeutic antibody by reducing its affinity for a transcytosis target” 2011) disclsoes a bispecific therapeutic antibody with a low affinity for TfR and a high affinity for the enzyme beta-secretase, an Alzheimer’s disease target.
Bohrmann (US 2019/0276530) discloses a blood brain barrier shuttle that includes a brain effector entity, a linker and one monovalent binding entity which binds to a blood brain barrier reeptor, wherein the linker couples the effector entity to the monovalent binding entity which binds to the blood brain barrier receptor. In particular embodiments, the monovalent binding entity which binds to the blood brian barrier receptor includes a blood brain barrier receptor ligand, scFv, Fv, sFab, VHH. In a particular emobidment, the monovalent binding entity is a scFab directed agasint the transferrin receptor.
Aptamers as Drug Delivery Vehicles:
Aptamers are single stranded DNA or RNA oligonucleotides that bind with high affinity to specific target molecules by folding into unique three-dimensional structures. The process of aptamer selection involves repeated steps of incubation of a high complexity library with a target of interest. Franciscis “Aptamer-Mediated Targeted Delivery of Therapeutics: An Update” Pharmaceuticals, 2016)
As aptamers function by molecular recognition, they can be developed for therapeutic applications with the same intended function as antibodies, such as drug delivery vehicles. Though analogous to their protein counterparts in regards to target recognition and application, aptamers possess numerous key advantages over antibodies and antibody fragments, the main being their size, pro- duction process and cost, stability and nucleic acid structure Shigdar, “Development of transferrin receptor aptamers as drug delivery vehicles for the treatment of brain metastases” Aptamers, 2018, Vol 2, 15–27).
Aptamers have been covalently or physically functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic functionalized with therapeutic compounds including chemotherapeutics, toxins, and therapeutic oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization oligonucleotides (siRNAs, miRNAs or antimiRs) and many methods of aptamer functionalization have been proposed as valid means to improve the specificity of nanoparticles. These approaches have been proposed as valid means to improve the specificity of nanoparticles. Franciscis “Aptamer-Mediated Targeted Delivery of Therapeutics: An Update” Pharmaceuticals, 2016)
Against Transferrin Receptor:
Wilner, (W O 2013/163303) discloses nuclease-stabilized aptamers which bind the human transferrin receptor and are readily internalized by cells which provides a means to specifically deliver cargoes to human cells which express this receptor.
Rossi (WO 2019/033051) discllsoes ribonucleic acid compound that includes RNA sequence capable of binding to a transferrin receptor (TfR). The compound may further include a compound moeity such as a therapeutic moiety attached to the RNA sequence. The compound/therapeutic moeity can be covalently attached to the RNA sequence. The therapeutic moiety can be an anticancer therapeutic moeity. Anticancer agents include monoclonal anitobdies such as anti-CD20, anti-HER2, anti-CD52. The ribonucleic acid compounds may be used to deliver compound moieties or therapetuic agents. In some embodimetns the nucleic acid compunds include an additional aptamer molecule such that a bispecific apatamer is formed. Preferably the additional aptamer does not target and/or bind to TfR.
Tan “Enhanced in vivo blood-brain barrier penetration by circular Tau-Transferrin receptor bifuncitonal aptamer for Tauopathy therapy” J. American Chemical Society, 2020, 142, 3862-3872) discoses a circular Tau-TfR bifunctional aptamer.
