Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain

Abstract

Alzheimer's disease (AD) and treatment of the brain in aging require the development of new biologic drugs, such as recombinant proteins or gene therapies. Biologics are large molecule therapeutics that do not cross the blood-brain barrier (BBB). BBB drug delivery is the limiting factor in the future development of new therapeutics for the brain. The delivery of recombinant protein or gene medicines to the brain is a binary process: either the brain drug developer re-engineers the biologic with BBB drug delivery technology, or goes forward with brain drug development in the absence of a BBB delivery platform. The presence of BBB delivery technology allows for engineering the therapeutic to enable entry into the brain across the BBB from blood. Brain drug development may still take place in the absence of BBB delivery technology, but with a reliance on approaches that have rarely led to FDA approval, e.g., CSF injection, stem cells, small molecules, and others. CSF injection of drug is the most widely practiced approach to brain delivery that bypasses the BBB. However, drug injection into the CSF results in limited drug penetration to the brain parenchyma, owing to the rapid export of CSF from the brain to blood. A CSF injection of a drug is equivalent to a slow intravenous (IV) infusion of the pharmaceutical. Given the profound effect the existence of the BBB has on brain drug development, future drug or gene development for the brain will be accelerated by future advances in BBB delivery technology in parallel with new drug discovery.

Keywords: Alzheimer’s disease; Trojan horse; blood-brain barrier; cerebrospinal fluid; endothelium.

Figure 1

Model for combination therapy of Alzheimer’s disease (AD) with blood-brain barrier (BBB)-penetrating biologic drugs is based on blocking the pathway leading to dementia at multiple levels within the brain. Pro-inflammatory cytokines in the brain, such as tumor necrosis factor (TNF)-α, cause neuro-inflammation, and this inflammation may accelerate Abeta amyloid peptide plaque formation. Plaque formation causes neurite dystrophy, leading to dementia. BBB-penetrating biologic drugs are re-engineered as IgG Trojan horse fusion proteins. Biologic TNF inhibitors, such as TNFα decoy receptors or anti-TNFα therapeutic antibodies, are respectively re-engineered as a BBB penetrating IgG-decoy receptor fusion protein or as a bispecific antibody. Therapeutic antibodies that disaggregate the Abeta amyloid plaque are re-engineered as Trojan horse bispecific antibodies. Neurotrophins are re-engineered as IgG-neurotrophin fusion proteins (Figure 4).

Figure 2

Whole-brain autoradiography study shows the limited penetration of biologic drugs into brain parenchyma following drug injection into the lateral ventricle (LV) in the rat. [¹²⁵I]-brain derived neurotrophic factor (BDNF) was injected into the LV and the brain removed 20 h later for autoradiography. The BDNF moves from the LV to the third ventricle (3V), then to the fourth ventricle, over the surface of the brain, and is absorbed into the blood of the superior sagittal sinus. The drug only distributes into ~0.2 mm of brain parenchyma ipsilateral to the injection with no measurable distribution to the contralateral brain. Reprinted by permission from Yan et al. (1994).

Figure 3

Endogenous BBB transporters include carrier-mediated transport (CMT) systems for certain small-molecule nutrients and receptor-mediated transport (RMT) systems for certain large molecule peptides or plasma proteins. (A) CMT systems include different members of the Solute Carrier (SLC) gene family, such as the GLUT1 glucose transporter for glucose and certain other hexoses, the MCT1 monocarboxylic acid transporter for lactate, pyruvate, and ketone bodies, the LAT1 large neutral amino acid transporter for phenylalanine (Phe) and over 10 other neutral amino acids, the CAT1 cationic amino acid transporter for arginine, lysine, and ornithine, the choline transporter, which may be the choline transporter-like protein-1 (CTL1), the CNT2 sodium-dependent purine nucleoside transporter for adenosine, guanosine, and inosine, and the nucleobase transporter (NBT) for purine bases such as adenine. (B) RMT systems include the insulin receptor (IR), the type 1 transferrin receptor (TfR1), the insulin-like growth factor receptor (IGFR), the leptin receptor (LEPR), the low-density lipoprotein receptor (LDLR), the neonatal Fc receptor (FcRn), and the LDLR related protein-1 (LRP1). Reprinted by permission from Pardridge (2017).

Figure 4

BBB transport of biologic drugs is enabled following the re-engineering of the biologic as an IgG fusion protein. The IgG domain acts as a molecular Trojan horse, and the human insulin receptor (HIR) monoclonal antibody (MAb) is used as the BBB Trojan horse for humans (Giugliani et al., 2018). If the biologic is a lysosomal enzyme, then the drug is re-engineered as a HIRMAb-enzyme fusion protein, which undergoes IR-mediated transport across the BBB, followed by IR-mediated endocytosis into the neuron. If the biologic is a neurotrophin, the drug is re-engineered as a HIRMAb-neurotrophin fusion protein, which undergoes IR-mediated transport across the BBB to enable binding of the neurotrophin domain of the fusion protein to the specific neuronal neurotrophin receptor (NTR). If the biologic is a decoy receptor, such as the extracellular domain (ECD) of the TNFα receptor, the drug is re-engineered as a HIRMAb-decoy receptor, which is transported across the BBB via the IR to enable sequestration within brain of TNFα. If the biologic is a therapeutic antibody, e.g., against the Abeta amyloid of AD, then the therapeutic antibody and the HIRMAb are re-engineered as a BBB-penetrating bispecific antibody, which traverses the BBB via the IR followed by engagement of the amyloid plaque in brain extracellular spaces. Reprinted by permission from Pardridge (2015a).

Figure 5

Autoradiography of serial sagittal sections of the Rhesus monkey brain obtained 2 h after the intravenous (IV) administration of either [¹²⁵I]-iduronidase (IDUA), a lysosomal enzyme, or [¹²⁵I]-HIRMAb-IDUA fusion protein. The brain uptake of the IDUA alone is minimal (A), whereas there is robust brain uptake of the HIRMAb-IDUA fusion protein (B). Panels (A,B) reprinted by permission from Boado and Pardridge (2017). There is higher uptake of the fusion protein in gray matter, as compared to white matter, owing to the greater vascular density in gray matter (Pardridge et al., 1995).

Figure 6

(A) Structure of a Trojan horse liposome (THL). A single plasmid DNA molecule is encapsulated in the interior of a ~100 nm liposome, the surface of which is conjugated with several thousand strands of 2,000 Da polyethylene glycol (PEG). The tips of 1%–2% of the PEG strands are conjugated with a receptor-specific monoclonal antibody (MAb). The MAb against either the IR or the transferrin receptor (TfR) engages the IR or TfR on the BBB to mediate transport into the brain and then binds the IR or TfR on the brain cells to trigger receptor-mediated endocytosis into brain cells. Panel (A) reprinted by permission from Pardridge (2002). (B) Electron micrograph of a THL complexed with a conjugate of a secondary antibody and 10 nm gold. The gold particles are about the same size as the PEG-extended MAb on the surface of the THL. Panel (B) reprinted by permission from Zhang et al. (2003a).

Figure 7

(A) Coronal section of Rhesus monkey brain after beta-galactosidase (LacZ) histochemistry. The primate brain was removed 2 days after the IV administration of a HIRMAb-targeted THL encapsulating a LacZ expression plasmid DNA. Magnification bar = 3 mm. (B) LacZ histochemistry of Rhesus monkey brain not injected with THLs. (C) Serial sections through the primate brain show the global distribution of the transgene in all parts of the primate brain following THL administration of the LacZ gene. Light microscopy of choroid plexus (D), occipital cortex (E), and cerebellum (F) after THL administration of the LacZ gene. Panels (A–F) reprinted by permission from Zhang et al. (2003c).

Figure 8

(A) Tyrosine hydroxylase (TH) immunohistochemistry (IHC) of rat brain removed 3 days after the IV administration of TfRMAb-targeted THLs encapsulating a rat TH expression plasmid DNA, under the influence of a human glial fibrillary acidic protein (GFAP) promoter, and 3.5 weeks after the unilateral injection of 8 μg of 6-hydroxydopamine into the right medial forebrain bundle. The injection dose of plasmid DNA was 10 μg/rat. (B) TH IHC of rat brain removed 3 days after the IV administration of THLs encapsulating the GFAP-TH transgene, but targeted only with a mouse IgG2a isotype control antibody with no specificity for a BBB receptor, and 3.5 weeks after the unilateral injection of 8 μg of 6-hydroxydopamine into the right medial forebrain bundle. There is a >90% loss of immunoreactive TH in the caudate-putamen nucleus (CPN) ipsilateral to toxin injection, and this loss is completely restored in the rats treated with the THL targeted with the TfRMAb (panel A), but not with the mouse IgG2a isotype control (panel B). (C) Confocal microscopy of the CPN region of the brain corresponding to panel (A), and TH immunostaining is shown in the red channel and immunostaining of neuN, a neuronal marker, is shown in the green channel. (D) Confocal microscopy of the CPN region of the brain corresponding to panel (B), and TH immunostaining is shown in the red channel and immunostaining of neuN is shown in the green channel. Magnification bar in panel (D) is 20 μm. Panels (A–D) reprinted by permission from Zhang et al. (2004a). (E) Apomorphine (left panel) and amphetamine (right panel)-induced rotation behavior in rats at 1 through 6 weeks after the administration of 8 μg of 6-hydroxydopamine in the right medial forebrain bundle. The rats were treated at weeks 1, 2, and 3 after intra-cerebral toxin administration with either saline or with TfRMAb-targeted THLs encapsulating a human prepro glial-derived neurotrophic factor (GDNF) transgene. The GDNF gene was under the influence of an 8 kb tissue-specific promoter taken from the 5′-flanking sequence of the rat TH gene. The differences in rotation in the THL and saline-treated rats are statistically significant at weeks 3, 4, 5, and 6 (*p < 0.05, ^‡p < 0.005, ^†p < 0.0005). Panel (E) reprinted by permission from Zhang and Pardridge (2009).

Figure 9

(A) Coronal section of the brain of severe combined immunodeficient (SCID) mouse at expiration following the implantation of 500,000 human U87 glioma cells in the caudate-putamen nucleus (CPN), and immunostained with the 528 monoclonal antibody (MAb) against the human epidermal growth factor receptor (EGFR). Panel (A) reprinted by permission from Zhang et al. (2002). (B) Section of SCID mouse brain at expiration after the implantation of 500,000 human U87 glioma cells in the CPN, and immunostained with the 8D3 MAb against the mouse transferrin receptor (TfR). The brain tumor-bearing mice were treated with weekly injections of saline starting 5 days after tumor implantation. Capillaries originating from normal mouse brains are seen vascularizing the human U87 tumor (*). (C) Survival of SCID mice following the implantation of 500,000 human U87 glioma cells in the CPN at day 0. Starting at 5 days after tumor implantation, the mice were treated with weekly IV injections of either saline or THLs doubly targeted with the 8D3 MAb against the mouse TfR, and the 83–14 MAb against the HIR. The THLs encapsulated a plasmid DNA that encoded a 29 nucleotide (nt) short interfering RNA (shRNA) that targeted nucleotides 2,529–2,557 of the human EGFR mRNA. The plasmid expression of the shRNA leads to RNA interference (RNAi) of the EGFR transcript in the brain tumor. The weekly injection dose of plasmid DNA was 5 μg/mouse. (D) Section of SCID mouse brain at expiration after the implantation of 500,000 human U87 glioma cells in the CPN, and immunostained with the 8D3 MAb against the mouse transferrin receptor (TfR). Starting at 5 days after tumor implantation, the mice were treated with weekly injections of the THLs doubly targeted with the TfRMAb and HIRMAb and encapsulating the plasmid DNA encoding the human EGFR mRNA-specific shRNA. There is an 80% reduction in capillary density in the tumor of the RNAi treated mice, as compared to the saline-treated mice (panel B). Panels (B–D) reprinted by permission from Zhang et al. (2004b).