Systemically Administered, Target-Specific, Multi-Functional Therapeutic Recombinant Proteins in Regenerative Medicine

Abstract

Growth factors, chemokines and cytokines guide tissue regeneration after injuries. However, their applications as recombinant proteins are almost non-existent due to the difficulty of maintaining their bioactivity in the protease-rich milieu of injured tissues in humans. Safety concerns have ruled out their systemic administration. The vascular system provides a natural platform for circumvent the limitations of the local delivery of protein-based therapeutics. Tissue selectivity in drug accumulation can be obtained as organ-specific molecular signatures exist in the blood vessels in each tissue, essentially forming a postal code system ("vascular zip codes") within the vasculature. These target-specific "vascular zip codes" can be exploited in regenerative medicine as the angiogenic blood vessels in the regenerating tissues have a unique molecular signature. The identification of vascular homing peptides capable of finding these unique "vascular zip codes" after their systemic administration provides an appealing opportunity for the target-specific delivery of therapeutics to tissue injuries. Therapeutic proteins can be "packaged" together with homing peptides by expressing them as multi-functional recombinant proteins. These multi-functional recombinant proteins provide an example how molecular engineering gives to a compound an ability to home to regenerating tissue and enhance its therapeutic potential. Regenerative medicine has been dominated by the locally applied therapeutic approaches despite these therapies are not moving to clinical medicine with success. There might be a time to change the paradigm towards systemically administered, target organ-specific therapeutic molecules in future drug discovery and development for regenerative medicine.

Keywords: angiogenesis; cell penetrating peptide; decorin; fibrosis; targeted delivery; transforming growth factor-β (TGF-β), bystander effect, CendR peptide, tissue regeneration, regenerative medicine, hypoxia, neuropilin-1, stem cell; vascular heterogeneity; vascular homing peptide.

Figure 1

The vascular zip codes. Each tissue has its own unique molecular structure in the luminal side of its vessels. These molecules function as receptors for tissue homing molecules such as peptides. Combining a homing peptide with another molecule creates a compound that can home to its target tissue. Homing diminishes the amount of the drug needed and helps to avoid side effects of the medication.

Figure 2

Target organ-specific delivery of therapeutics in regenerative medicine. The angiogenic blood vessels that form in regenerating tissues after injury are structurally and molecularly different from the dormant blood vessels elsewhere in the body. They provide an accessible and abundant target for the organ-specific delivery of therapeutics during the repair of the tissue injuries. They can be targeted by vascular homing peptides that bind to their receptor selectively expressed only in angiogenic blood vessels. Drugs administered systemically can be converted to target tissue-specific by coupling them to the vascular homing peptide.

Figure 3

Mechanism of action of multi-functional re-engineered decorin in inhibition of scar formation. Schematic presentation on recombinant fusion protein consisting of decorin (DCN) and the vascular homing and cell penetrating peptide CAR. CAR-decorin ① is a multifunctional biotherapeutic that inhibits numerous growth factor signaling pathways involved in fibrosis. The systemically administered molecule is targeted by CAR peptide to the inflammatory or angiogenic vasculature in any organ of the body ②. The CAR homing peptide binds to its receptor (“zip code”) in angiogenic or inflammatory vasculature ② and as cell penetrating peptide, it delivers the fusion molecule deep in the target organ parenchyma ③. The CAR peptide then binds to heparin sulfate proteoglycans on the cell surface of the stromal cells ③. This binding facilitates the neutralization of scar forming isoforms TGF-β1 and TGF-β2 by the therapeutic part of the molecule, DCN ④. This mechanism results in a therapeutic response, i.e. reduction of scar formation in skin wound. Picture by Helena Schmidt (With permission from Suomalainen Lääkäriseura Duodecim 2011).

Figure 4

The bystander effect enables delivering a therapeutic into the tissue without a covalent link between the therapeutic and the homing peptide. The drawing represents the homing of a peptide (diamond) to its target tissue (cuboidal cells) and the bystander effect created by the peptide. The tissue on the right has the receptor for the homing peptide (Y-shape). When the peptide binds to its receptor, it opens a pathway for the therapeutic molecule (triangle) to pass through the vascular wall endothelium and enter the tissue parenchyma.

Figure 5

The function of the C-end Rule sequence. Peptides containing the C-end Rule (CendR) sequence are capable of penetrating to tissues and delivering cargo. Typically, the CendR sequence is cryptic and needs to be exposed. The receptor specific for the target tissue (fork) first binds the homing peptide enabling its cleavage by an enzyme (blue), which results in the exposure of the CendR sequence (RXXR). The CendR sequence then binds to another receptor (half a circle), neuropilin-1 (NRP-1), which triggers the cell membrane to form a vesicle containing the peptide and other molecules close to it. The vesicle is then transferred through the cell to let its contents enter to the tissue parenchyma.

Figure 6

Vascular homing peptide induced tissue-selective vasodilation in pulmonary arterial hypertension (PAH). (A) Vascular homing and cell penetrating peptide CAR homes to pulmonary arteries in disease-specific fashion in experimental models of PAH [27,28]. A little bit of control peptide (mutant CAR peptide) binding can be seen to similar blood vessel in PAH. (B) Effect of CAR (0.3 mg/kg) and Rho-kinase inhibitor Y27632 (1 mg/kg) mixture on right ventricle (RVSP, right ventricle systolic pressure) and left ventricle (SAP, systemic arterial pressure) systolic pressure in PAH. The CAR/Y27632 combination treatment induced a marked pulmonary-specific vasodilation RVSP with only a minimum effect on SAP in PAH. (C) Schematic presentation of the “bystander effect”, i.e. target organ-specific drug delivery, in PAH. Reproduced from [28] with permission from Elsevier 2017.