RECOMBINANT HEPARAN SULFATES

TECHNICAL FIELD

The present disclosure relates broadly to molecular biology. In particular, the present disclosure relates to recombinant heparan sulfates.

BACKGROUND

Human mesenchymal stem/stromal cells (hMSCs) are promising candidates for use in regenerative therapy due to their propensity to secrete trophic and immunomodulatory factors. MSCs isolated from bone marrow aspirate and other sources must go through extensive ex vivo expansion to generate the large number of cells which constitute a therapeutic dose. Supplementing the media with growth factors, such as fibroblast growth factor 2 (FGF2), has been shown to be effective in enhancing the hMSC proliferation in ex vivo cultures. However, prolonged supplementation of FGF2 not only affects the sternness and potency of hMSC but also increases the overall cost of the cell culture media. One potential alternative to the use of FGF2 as a MSC media additive is heparan sulfate (HS). Heparan sulfate is a highly heterogeneous and negatively charged linear polysaccharide comprising of repeating units of glucuronic acid and N- acetylglucosamine. Heparan sulfate plays essential roles in cell survival and homeostasis by acting as a co-receptor for several growth factors, morphogens, cytokines and chemokines. Addition of heparan sulfate into culture media has been shown to improve the hMSC maintenance and growth by enhancing FGF2 stability, bioavailability, and bioactivity. Heparan sulfate has also displayed potential in several other applications, including enhancing bone repair, stimulating angiogenesis and as a potential inhibitor of multiple infectious diseases.

The biosynthesis of heparan sulfate in cells is an intricate and highly complex process. Heparan sulfate biosynthesis begins with the synthesis of heparan sulfate proteoglycan core proteins (HSPG) in the endoplasmic reticulum. Well-studied HSPGs include serglycin, perlecan, the syndecans and the glypicans. After synthesis, HSPGs are then transported to the Golgi where a tetrasaccharide linker (glucuronic acid- galactose-galactose-xylose) is attached to serine residues within the HSPG. This tetrasaccharide can be further modified by the co-polymerases exostosin 1 and 2, giving rise to a nascent heparan sulfate chain. The modification of heparan sulfate involves several families of enzymes with multiple isoforms, each with different substrate specificities, working in succession. The first modification event is performed by enzymes of the N-deacetylase/N-sulfotransferase (NDST) family, which catalysed the N- deacetylation and N-sulfation of selected N-acetylglucosamine residues. After N- sulfation, Glucuronyl C5-epimerase (GLCE) catalysed the C5 epimerization of glucuronic acid residues to L-iduronic acid. 2-O-sulfation of iduronic acid and some glucuronic acid is carried out by 2-O-sulfotransferase (2OST) catalysed the O-sulfation of uronic acids, while 6-O-sulfotransferase (6OST) and 3-O-sulfotransferase (3OST) catalysed the O- sulfation of glucosamine residues. The modification of heparan sulfate is template independent, and the final structure and composition of heparan sulfate is dependent on numerous factors, including the relative abundance of each biosynthetic enzyme within the Golgi. Therefore, heparan sulfate structure and composition can be influenced through metabolic engineering, by either knocking out or overexpressing any of the various biosynthetic enzymes or HSPG core proteins. Alterations in the structure of heparan sulfate will influence binding interactions between heparan sulfate and heparan sulfate binding proteins, thereby altering its effect on different cell types.

Traditional approaches to mammalian cell engineering rely on the transfection of cells with plasmid vectors and subsequent selection of stable transfectants using antibiotics or metabolic enzyme inhibitors. Owing to the nature of random integration and variation in copies of the transgenes that are integrated into the genome, the stably transfected pool consists of cells that are highly heterogeneous in expression level and stability. Time-consuming and laborious screening of clones must be carried out to achieve desirable expression characteristics. As such, the number of genes that can be engineered is limited and little success has been obtained for engineering complex metabolic pathways involving multiple enzymes, such as the heparan sulfate biosynthesis pathway. Previous studies in the art were successful in engineering two genes simultaneously, and more recently a recent study in the art successfully engineer three genes simultaneously in murine mastocytoma cells.

Most commercially available heparan sulfate is isolated as a side fraction during the manufacturing of pharmaceutical-grade heparin from porcine intestinal mucosa. Whilst heparan sulfate has displayed promise in numerous applications, its animal- derived nature prohibits its use in many therapeutic applications. In addition, low availability of porcine intestinal mucosa, high cost of production and batch -to- batch variability in structure, composition and activity have hindered the use of heparan sulfate in wider applications. These issues highlight the importance of moving away from animal- derived heparan sulfate products to the one that can be generated under controlled conditions.

In view of the above, there is a need to provide an alternative source of heparan sulfate. There is a need to provide recombinant heparan sulfates.

SUMMARY

In one aspect, there is provided a nucleic acid construct comprising one or more internal ribosome entry site (IRES), one or more gene of interest, and a pair of site-specific recombinase, wherein the gene of interest is a heparan sulfate variant gene, a heparan sulfate modification enzyme gene, and/or a heparan sulfate scaffold protein gene.

In some examples, the site-specific recombinase flanks both ends of the nucleic acid construct.

In some examples, the site-specific recombinase is selected from the group consisting of FLPe, heterologous FRT3 and/or FRT, Cre-LoxP, and BXB1-attP-attB.

In some examples, the nucleic acid construct further comprises a cap, optionally the nucleic acid construct comprises a polyA tail.

In some examples, the gene of interest comprises one or more of the following genes:

In some examples, the heparan sulfate scaffold protein gene is a heparan sulfate proteoglycan gene. In some examples, the heparan sulfate proteoglycan gene is selected from the group consisting of SRGN, GPC1 , HSPG2, and SDC1.

In some examples, the heparan sulfate modification enzyme gene is selected from the group consisting of N-Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-O-Sulfotransferase 1 (HS2ST7)/2OST1 , HS3ST1/3OST1 , HS3ST5/3OST5, HS6ST1/6OST1 ,

HS6ST2/6OST2, and HS6ST3/6OST3. In some examples, the construct further comprises a selection gene, optionally wherein the selection gene is an antibiotic resistant gene.

In some examples, the selection gene comprises one or more of a hygromycin resistant gene or puromycin resistant gene.

In some examples, the IRES is a wild type IRES.

In some examples, the construct is selected from the group consisting of one or more gene of interest, two or more genes of interest, three or more genes of interest, four or more genes of interest, or one gene of interest, or two genes of interest, or three genes of interest, or four genes of interest.

In some examples, the construct comprises: one or more IRES, one gene of interest, and a pair of site-specific recombinase; or one or more IRES, two genes of interest, and a pair of site-specific recombinase; or one or more IRES, three genes of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, one gene of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, two genes of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, three genes of interest, and a pair of site-specific recombinase; wherein optionally the construct further comprises a polyA tail.

In some examples, the construct comprises: a selection gene, an IRES, a heparan sulfate scaffold protein gene, and a pair of site-specific recombinase; or a selection gene, an IRES, a heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or a selection gene, one or more IRES, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or a selection gene, one or more IRES, one or more heparan sulfate scaffold protein gene, one or more heparan sulfate modification enzyme gene, and a pair of sitespecific recombinase; or a selection gene, one or more IRES, a heparan sulfate scaffold protein gene, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or an IRES, a heparan sulfate scaffold protein gene, and a pair of site-specific recombinase; or an IRES, a heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or one or more IRES, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or one or more IRES, one or more heparan sulfate scaffold protein gene, one or more heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or one or more IRES, a heparan sulfate scaffold protein gene, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase, wherein optionally the construct further comprises a polyA tail.

In some examples, the construct comprises:

- a selection gene, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, and a pair of site-specific recombinase; and/or - a selection gene, IRES, SERGLYCIN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, PERLECAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, SYNDECAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLYPICAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, GLCE, and a pair of site-specific recombinase; and/or - a selection gene, IRES, GLCE, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST2, and a pair of site-specific recombinase; and/or - a selection gene, IRES, GLCE, IRES, HS2ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS3ST1, and pair of site-specific recombinase; and/or - a selection gene, IRES, GLCE, IRES, HS3ST5, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS6ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS6ST1, and pair of site-specific recombinase; and/or - a selection gene, /RES, GLCE, /RES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, /RES, HS6ST3, IRES, HS6ST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, /RES, NDST2, (RES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS6ST3, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS6ST3, and pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST3, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein gene, an IRES, GLCE, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, NDST2, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS2ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS3ST5, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein gene, an IRES, HS6ST2, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS3ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- HSPG2, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- HSPG2, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or

- HSPG2, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or - HSPG2, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or

- SDC1, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase, optionally wherein the construct further comprises a poly A tail.

In some examples, the nucleic acid construct further comprises a promoter, optionally wherein the promoter is for expressing a heparan sulfate scaffold protein.

In some examples, the promoter is a chimeric promoter (ChiP).

In some examples, the nucleic acid construct is a vector, optionally a landing pad vector.

In some examples, the nucleic acid construct as disclosed herein.

In another aspect, there is provided a host cell and/or cell line comprising the nucleic acid construct as disclosed herein, optionally the host cell is a Chinese Hamster Ovary (CHO) cell line. In yet another aspect, there is provided a recombinant heparan sulfate comprising a selection protein, and a heparan sulfate scaffold protein and/or a heparan sulfate modification enzyme; or a selection protein, two heparan sulfate modification enzyme; or a selection protein, one or more heparan sulfate scaffold protein, and one or more heparan sulfate modification enzyme; or a selection protein, a heparan sulfate scaffold protein, and two heparan sulfate modification enzymes; or a heparan sulfate scaffold protein and/or a heparan sulfate modification enzyme; or two heparan sulfate modification enzymes; or one or more heparan sulfate scaffold protein gene, and one or more heparan sulfate modification enzyme; or a heparan sulfate scaffold protein gene, and two heparan sulfate modification enzyme.

In some examples, the protein comprises:

- a selection protein, and GLCE; and/or

- a selection protein, and NDST1 ; and/or

- a selection protein, and NDST2; and/or

- a selection protein, and HS2ST1 ; and/or

- a selection protein, and HS3ST1 ; and/or

- a selection protein, and HS3ST5; and/or

- a selection protein, and HS6ST1 ; and/or

- a selection protein, and HS6ST2; and/or

- a selection protein, and HS6ST3; and/or

- a selection protein, and serglycin; and/or

- a selection protein, and perlecan; and/or

- a selection protein, and syndecan; and/or - a selection protein, and glypican; and/or

- a selection protein, GLCE, and NDST2; and/or

- a selection protein, HS6ST1, and NDST2; and/or

- a selection protein, HS6ST2, and NDST2; and/or

- a selection protein, HS6ST3, and NDST2; and/or

- a selection protein, NDST1, and GLCE; and/or

- a selection protein, NDST2, and GLCE; and/or

- a selection protein, HS2ST1, and GLCE; and/or

- a selection protein, HS3ST1, and GLCE; and/or

- a selection protein, HS3ST5, and GLCE; and/or

- a selection protein, HS6ST1, and GLCE; and/or

- a selection protein, HS6ST2, and GLCE; and/or

- a selection protein, HS6ST3, and GLCE; and/or

- a selection protein, GLCE, and NDST1; and/or

- a selection protein, NDST2, and NDST1; and/or

- a selection protein, HS2ST1, and NDST1; and/or

- a selection protein, HS3ST1, and NDST1; and/or

- a selection protein, HS3ST5, and NDST1; and/or

- a selection protein, HS6ST1, and NDST1; and/or

- a selection protein, HS6ST2, and NDST1; and/or

- a selection protein, HS6ST3, and NDST1; and/or

- a selection protein, GLCE, and NDST2; and/or

- a selection protein, NDST1, and NDST2; and/or

- a selection protein, HS2ST1, and NDST2; and/or

- a selection protein, HS3ST1, and NDST2; and/or

- a selection protein, HS3ST5, and NDST2; and/or

- a selection protein, HS6ST1, and NDST2; and/or

- a selection protein, HS6ST2, and NDST2; and/or

- a selection protein, HS6ST3, and NDST2; and/or

- a selection protein, GLCE, and HS2ST1; and/or - a selection protein, NDST1, and HS2ST1; and/or

- a selection protein, NDST2, and HS2ST1; and/or

- a selection protein, HS3ST1, and HS2ST1; and/or

- a selection protein, HS3ST5, and HS2ST1; and/or

- a selection protein, HS6ST1, and HS2ST1; and/or

- a selection protein, HS6ST2, and HS2ST1; and/or

- a selection protein, HS6ST3, and HS2ST1; and/or

- a selection protein, GLCE, and HS3ST1; and/or

- a selection protein, NDST1, and HS3ST1; and/or

- a selection protein, NDST2, and HS3ST1; and/or

- a selection protein, HS2ST1, and HS3ST1; and/or

- a selection protein, HS3ST5, and HS3ST1; and/or

- a selection protein, HS6ST1, and HS3ST1; and/or

- a selection protein, HS6ST2, and HS3ST1; and/or

- a selection protein, HS6ST3, and HS3ST1; and/or

- a selection protein, GLCE, and HS3ST5; and/or

- a selection protein, NDST1, and HS3ST5; and/or

- a selection protein, NDST2, and HS3ST5; and/or

- a selection protein, HS2ST1, and HS3ST5; and/or

- a selection protein, HS3ST1 , and HS3ST5; and/or

- a selection protein, HS6ST1, and HS3ST5; and/or

- a selection protein, HS6ST2, and HS3ST5; and/or

- a selection protein, HS6ST3, and HS3ST5; and/or

- a selection protein, GLCE, and HS6ST1; and/or

- a selection protein, NDST1 , and HS6ST1; and/or

- a selection protein, NDST2, and HS6ST1; and/or

- a selection protein, HS2ST1, and HS6ST1; and/or

- a selection protein, HS3ST1, and HS6ST1; and/or

- a selection protein, HS3ST5, and HS6ST1; and/or

- a selection protein, HS6ST2, and HS6ST1; and/or

- a selection protein, HS6ST3, and HS6ST1; and/or - a selection protein, GLCE, and HS6ST2; and/or

- a selection protein, NDST1, and HS6ST2; and/or

- a selection protein, NDST2, and HS6ST2; and/or

- a selection protein, HS2ST1, and HS6ST2; and/or

- a selection protein, HS3ST1, and HS6ST2; and/or

- a selection protein, HS3ST5, and HS6ST2; and/or

- a selection protein, HS6ST1, and HS6ST2; and/or

- a selection protein, HS6ST3, and HS6ST2; and/or

- a selection protein, GLCE, and HS6ST3; and/or

- a selection protein, NDST1, and HS6ST3; and/or

- a selection protein, NDST2, and HS6ST3; and/or

- a selection protein, HS2ST1, and HS6ST3; and/or

- a selection protein, HS3ST1, and HS6ST3; and/or

- a selection protein, HS3ST5, and HS6ST3; and/or

- a selection protein, HS6ST1, and HS6ST3; and/or

- a selection protein, HS6ST2, and HS6ST3; and/or

- a HS scaffold protein, NDST1 , and GLCE; and/or

- a HS scaffold protein, NDST2, and GLCE; and/or

- a HS scaffold protein, HS2ST1 , and GLCE; and/or

- a HS scaffold protein, HS3ST1 , and GLCE; and/or

- a HS scaffold protein, HS3ST5, and GLCE; and/or

- a HS scaffold protein, HS6ST1 , and GLCE; and/or

- a HS scaffold protein, HS6ST2, and GLCE; and/or

- a HS scaffold protein, HS6ST3, and GLCE; and/or

- a HS scaffold protein, GLCE, and NDST1; and/or

- a HS scaffold protein, NDST2, and NDST1; and/or

- a HS scaffold protein, HS2ST1 , and NDST1 ; and/or

- a HS scaffold protein, HS3ST1 , and NDST1 ; and/or

- a HS scaffold protein, HS3ST5, and NDST1 ; and/or

- a HS scaffold protein, HS6ST1 , and NDST1; and/or

- a HS scaffold protein, HS6ST2, and NDST1 ; and/or

- a HS scaffold protein, HS6ST3, and NDST1 ; and/or - a HS scaffold protein, GLCE, and NDST2; and/or

- a HS scaffold protein, NDST1 , and NDST2; and/or

- a HS scaffold protein, HS2ST1 , and NDST2; and/or

- a HS scaffold protein, HS3ST1 , and NDST2; and/or

- a HS scaffold protein, HS3ST5, and NDST2 and/or

- a HS scaffold protein, HS6ST1 , and NDST2; and/or

- a HS scaffold protein, HS6ST2, and NDST2; and/or

- a HS scaffold protein, HS6ST3, and NDST2; and/or

- a HS scaffold protein, GLCE, and HS2ST1; and/or

- a HS scaffold protein, NDST1 , and HS2ST1; and/or

- a HS scaffold protein, NDST2, and HS2ST1 ; and/or

- a HS scaffold protein, HS3ST 1 , and HS2ST 1 ; and/or

- a HS scaffold protein, HS3ST5, and HS2ST1; and/or

- a HS scaffold protein, HS6ST 1 , and HS2ST 1 ; and/or

- a HS scaffold protein, HS6ST2, and HS2ST1 ; and/or

- a HS scaffold protein, HS6ST3, and HS2ST 1 ; and/or

- a HS scaffold protein, GLCE, and HS3ST1; and/or

- a HS scaffold protein, NDST1 , and HS3ST1; and/or

- a HS scaffold protein, NDST2, and HS3ST1 ; and/or

- a HS scaffold protein, HS2ST1 , and HS3ST1; and/or

- a HS scaffold protein, HS3ST5, and HS3ST 1 ; and/or

- a HS scaffold protein, HS6ST 1 , and HS3ST 1 ; and/or

- a HS scaffold protein, HS6ST2, and HS3ST1 ; and/or

- a HS scaffold protein, HS6ST3, and HS3ST 1 ; and/or

- a HS scaffold protein, GLCE, and HS3ST5; and/or

- a HS scaffold protein, NDST1 , and HS3ST1 ; and/or

- a HS scaffold protein, NDST2, and HS3ST5; and/or

- a HS scaffold protein, HS2ST 1 , and HS3ST5; and/or

- a HS scaffold protein, HS3ST 1 , and HS3ST5; and/or

- a HS scaffold protein, HS6ST 1 , and HS3ST5; and/or

- a HS scaffold protein, HS6ST2, and HS3ST5; and/or - a HS scaffold protein, HS6ST3, and HS3ST5; and/or

- a HS scaffold protein, GLCE, and HS6ST1; and/or

- a HS scaffold protein, NDST1 , and HS6ST1 ; and/or

- a HS scaffold protein, NDST2, and HS6ST1 ; and/or

- a HS scaffold protein, HS2ST 1 , and HS6ST 1 ; and/or

- a HS scaffold protein, HS3ST 1 , and HS6ST 1 ; and/or

- a HS scaffold protein, HS3ST5, and HS6ST 1 ; and/or

- a HS scaffold protein, HS6ST2, and HS6ST1 ; and/or

- a HS scaffold protein, HS6ST3, and HS6ST 1 ; and/or

- a HS scaffold protein, GLCE, and HS6ST2; and/or

- a HS scaffold protein, NDST1 , and HS6ST2; and/or

- a HS scaffold protein, NDST2, and HS6ST2; and/or

- a HS scaffold protein, HS2ST1 , and HS6ST2; and/or

- a HS scaffold protein, HS3ST1 , and HS6ST2; and/or

- a HS scaffold protein, HS3ST5, and HS6ST2; and/or

- a HS scaffold protein, HS6ST1, and HS6ST2; and/or

- a HS scaffold protein, HS6ST3, and HS6ST2; and/or

- a HS scaffold protein, GLCE, and HS6ST3; and/or

- a HS scaffold protein, NDST1 , and HS6ST3; and/or

- a HS scaffold protein, NDST2, and HS6ST3; and/or

- a HS scaffold protein, HS2ST1, and HS6ST3; and/or

- a HS scaffold protein, HS3ST 1 , and HS6ST3; and/or

- a HS scaffold protein, HS3ST5, and HS6ST3; and/or

- a HS scaffold protein, HS6ST 1 , and HS6ST3; and/or

- a HS scaffold protein, HS6ST2, and HS6ST3; and/or

- serglycin, GLCE, and NDST2; and/or

- serglycin, HS6ST1, and NDST2; and/or

- serglycin, HS6ST2, and NDST2; and/or

- serglycin, HS6ST3, and NDST2; and/or

- perlecan, GLCE, and NDST2; and/or - perlecan, HS6ST1, and NDST2; and/or

- perlecan, HS6ST2, and NDST2; and/or

- perlecan, HS6ST3, and NDST2; and/or

- syndecan, GLCE, and NDST2; and/or

- syndecan, HS6ST1, and NDST2; and/or

- syndecan, HS6ST2, and NDST2; and/or

- syndecan, HS6ST3, and NDST2; and/or

- glypican, GLCE, and NDST2; and/or

- glypican, HS6ST1, and NDST2; and/or

- glypican, HS6ST2, and NDST2; and/or

- glypican, HS6ST3, and NDST2.

In yet another aspect, there is provided a recombinant heparan sulfate as disclosed herein for use in enhancing cell proliferation or in enhancing stability of cytokine, growth factors, morphogens, chemokines, and/or receptors.

In yet another aspect, there is provided a method of culturing a cell and/or inducing a cell proliferation and/or expanding a cell, the method comprising culturing the cell in a media comprising the recombinant heparan sulfate as disclosed herein.

In yet another aspect, there is provided a method of generating a recombinant heparan sulfate comprising transfection of the vector as disclosed herein into a cell line.

In yet another aspect, there is provided a kit for generating a recombinant heparan sulfate comprising a nucleic acid construct as disclosed herein, and an instruction to perform the method as disclosed herein.

In yet another aspect, there is provided a kit for culturing a cell comprising a recombinant heparan sulfate as disclosed herein, and an instruction to perform the method of culturing a cell as disclosed herein. DESCRIPTION OF EMBODIMENTS

The use of human mesenchymal stem cells (hMSCs) in cell therapy requires multiple passaging to get the cell numbers required. Serial passaging of hMSCs in media containing growth factors such as FGF2 leads to loss of sternness and cell senescence. Heparan sulfates are polysaccharides that can bind FGF2 and its receptor and can be used as a media additive to support hMSC growth. In addition to FGF2, heparan sulfate can be used to stabilise a variety of other growth factors, morphogens, chemokines, and cytokines, including interleukin 2 (IL2), bone morphogenetic protein 2 (BMP2), vascular endothelial growth factor (VEGF) and transforming growth factor pi (TGFpi). This makes heparan sulfate suitable as a media additive for other therapeutically relevant cell types, such as T cells. However, heparan sulfate (HS) is not commercially available at large scale (10 mg+), limiting its use in numerous applications, including as a potent stem cell media bio-additive. Heparan sulfate that is available is primarily isolated from animal tissues, which is undesirable when striving to develop chemically defined, xeno-free products. To counter these issues, the inventors of the present disclosure have developed a method for producing recombinant heparan sulfate variants in vitro through the metabolic engineering of cells, tailored to possess enhanced affinity toward proteins of choice. These recombinant heparan sulfate variants can then be used in various applications, including as media bio-additives to enhance stem cell growth.

In one aspect, there is provided a nucleic acid construct comprising one or more internal ribosome entry site (IRES), one or more gene of interest, and a pair of sitespecific recombinases, wherein the gene of interest is a heparan sulfate variant gene, a heparan sulfate modification enzyme gene, and/or a heparan sulfate scaffold protein gene.

As used herein, a site-specific recombinase refers to a group of nucleic acid sequences that are capable of rearranging nucleic acid sequences by breaking and rejoining the strands at specific points. As such, when site-specific recombinase can facilitate specialized recombination that is an exchange between two different nucleic acid molecules. The exchange may be an integration, excision, or inversion of the nucleic acid molecules. Therefore, site-specific recombinase enables a targeted (or site-specific) insertion of a nucleic acid sequence into the genome of a cell.

Without wishing to be bound by theory, the use of site-specific recombinase in the construct as described herein allows the integration of a single copy of the construct as described herein into the genome of a cell line. In turn, this integration to a single spot in the genome ensures the elimination of the confounding effects that copy number variation and different integration sites have on gene expression. This is important as it allows for direct comparison of the impact that different modifications have on the yield and quality of heparan sulfate when tested against other gene combinations.

In addition, unlike protein synthesis, heparan sulfate synthesis in cells is template independent and relies on the relative amount of the different heparan sulfate modification enzymes within the Golgi to produce specific heparan sulfate structures. As such, the inventors of the present disclosure believe that multiple copy integration of the transgene may disadvantageously result in increased production of these enzymes within the cells, thereby changing the desired carbohydrate (such as heparan sulfate) structure further. In contrast to the constructs and/or methods as described herein, random integration methods commonly used in the art have no control over the copy number in the cell and will result in extensive clonal screening process downstream of the workflow as cells in the pools differs greatly from each other in terms of stability and productivity. Therefore, the inventors of the present disclosure solve the problem of random integration by providing constructs that allow for site-specific integration.

With regards to the integration site, many studies have shown that the integration site greatly influences transgene expression and is a factor that determines cell line stability. In any cell type, the majority of the genome consists of transcriptionally inactive heterochromatin. If transgenes are randomly integrated into the genome, there is a high possibility that the gene of interest will be integrated into an area which is not favorable for high level and stable expression. In the present disclosure, the transgene construct is targeted to a predefined ‘landing pad’ which has been validated to produce stable and high producing cells.

Compared to random integration approaches, the targeted integration approach directs the insertion of a transgene into a specific locus in the genome, solving the problem of heterogeneous gene expression and clonal variation issues faced from randomly inserted genes. Cells generated through the targeted integration approach have the same genetic background, resulting in cells exhibiting high phenotypic and genetic homogeneity. Recombinase mediated cassette exchange (RMCE) is widely used for generating targeted integration cell lines. The RMCE approach uses recombinases, which recognizes specific recombination sites pre-inserted into the genome and mediate the exchange of the regions between two incompatible sites with that of the transgene. Among the recombinases available, Flp/FRT is the most widely used recombinase for CHO cell engineering due to its high specificity, exchange efficiency and low cytotoxicity. Therefore, the inventors of the present disclosure hypothesize that by using targeted integration and combining the overexpression of HSPG core proteins and critical heparan sulfate biosynthetic enzymes, the inventors of the present disclosure can alter the structure, composition and increase the overall yield of heparan sulfate produced in CHO cells.

In some examples, the site-specific recombinase is a recombinase target site flank (or pair). In some examples, the site-specific recombinase may be a heterologous recombinase.

In some examples, wherein the site-specific recombinase flanks both ends of the nucleic acid construct.

In some examples, wherein the site-specific recombinase may include, but is not limited to, improved flippase recombinase (FLPe), heterologous flippase recognition target 3 (FRT3) and/or flippase recognition target (FRT), Cre-LoxP, BXB1-attP-attB, and the like.

In some examples, the FRT3 is composed of two 13bp inverted repeats flanking an 8bp asymmetric spacer sequence (5'-

GAAGTTCCTATTCttcaaataGTATAGGAACTTC-3’) (SEQ ID NO: 1) and contains mutations in the spacer sequence compared to FRT (PMID:7947678). In some examples, the FRT is composed of two 13bp inverted repeats flanking an 8bp asymmetric spacer sequence (5'-GAAGTTCCTATTCtctagaaaGTATAGGAACTTC- 3')(SEQ ID NO: 2).

In some examples, wherein the nucleic acid construct further comprises a cap, optionally the nucleic acid construct comprises a polyA tail.

In some examples, wherein the gene of interest comprises one or more of the following genes:

In some examples, wherein the heparan sulfate scaffold protein gene is a heparan sulfate proteoglycan gene.

In some examples, the heparan sulfate proteoglycan gene may be a mammalian heparan sulfate scaffold protein gene. In some examples, the heparan sulfate proteoglycan gene may be from human, non-human primates, pig, mouse, rat, and the like. In some examples, the heparan sulfate proteoglycan gene may include, but is not limited to SRGN, HSPG2, SDC1, GPC1, syndecan 2 (SDC2), syndecan 3 (SDC3), syndecan 4 (SDC4), glypican 2 (GPC2), glypican 3 (GPC3), glypican 4 (GPC4), glypican 5 (GPC5), glypican 6 (GPC6), and the like.

In some examples, wherein the heparan sulfate proteoglycan gene comprises SRGN, GPC1 , HSPG2 or SDCI .

In some examples, wherein the heparan sulfate modification enzyme gene comprises N- Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-0-Sulfotransferase 1 (/7S2ST7)/2OST1 , HS3ST1/3OST1, HS3ST5/3OST5, HS6ST1/6OST1 , HS6ST2/6OST2, or HS6ST3/6OST3.

In some examples, wherein the construct further comprises a selection gene.

In some examples, wherein the selection gene is an antibiotic resistant gene.

In some examples, wherein the selection gene comprises one or more of a hygromycin resistant gene, or puromycin resistant gene.

In some examples, the selection gene may be included downstream of the construct landing pad. For example, the selection may be based on the use of an ATG- less puromycin which sits downstream of the CHO landing pad. In such examples, integration of a gene construct (such as a triple gene construct) into the landing pad will place an ATG upstream of the inactive puromycin, thereby activating it for use in selection.

In some examples, wherein the IRES is a wild type IRES.

In some examples, the construct as described herein may comprise one IRES, two IRES, three IRES, or four IRES, or one or more IRES, or two or more IRES, or three or more IRES, or four or more IRES.

In some examples, the construct as described herein may comprise one or more promoters to drive the expression of the different genes. In some examples, the construct as described herein may comprise two promoters, three promoters, four promoters, or five promoters to drive the expression of the genes in the construct.

In some examples, wherein the construct comprises one or more gene of interest, two or more genes of interest, three or more genes of interest, four or more genes of interest, or one gene of interest, or two genes of interest, or three genes of interest, or four genes of interest, and the like.

In some examples, the nucleic acid construct may include a nucleic acid in the 5’ to 3’ direction or a protein from the N terminus to the C terminus.

In some examples, wherein the construct comprises: one or more IRES, one gene of interest, and a pair of site-specific recombinase; or one or more IRES, two genes of interest, and a pair of site-specific recombinase; or one or more IRES, three genes of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, one gene of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, two genes of interest, and a pair of site-specific recombinase; or a selection gene, an IRES, three genes of interest, and a pair of site-specific recombinase; wherein optionally the construct further comprises a polyA tail.

In some examples, wherein the construct comprises: a selection gene, an IRES, a heparan sulfate scaffold protein gene, and a pair of site-specific recombinase; or a selection gene, an IRES, a heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or a selection gene, one or more IRES, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or a selection gene, one or more IRES, one or more heparan sulfate scaffold protein gene, one or more heparan sulfate modification enzyme gene, and a pair of sitespecific recombinase; or a selection gene, one or more IRES, a heparan sulfate scaffold protein gene, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or an IRES, a heparan sulfate scaffold protein gene, and a pair of site-specific recombinase; or an IRES, a heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or one or more IRES, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase; or one or more IRES, one or more heparan sulfate scaffold protein gene, one or more heparan sulfate modification enzyme gene, and a pair of site-specific recombinase; or one or more IRES, a heparan sulfate scaffold protein gene, two heparan sulfate modification enzyme genes, and a pair of site-specific recombinase, wherein optionally the construct further comprises a polyA tail.

In some examples, wherein the construct comprises:

- a selection gene, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, SERGLYCIN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, PERLECAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, SYNDECAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLYPICAN, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, GLCE, and a pair of site-specific recombinase; and/or - a selection gene, IRES, NDST2, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, GLCE, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, NDST2, and a pair of site-specific recombinase; and/or - a selection gene, IRES, NDST1, IRES, NDST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, NDST2, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS2ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS3ST1, and pair of site-specific recombinase; and/or - a selection gene, IRES, NDST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS3ST1, and pair of site-specific recombinase; and/or - a selection gene, IRES, HS2ST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS3ST1, and pair of site-specific recombinase; and/or - a selection gene, IRES, HS6ST1, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS3ST1, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or - a selection gene, IRES, HS3ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS3ST5, and pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS6ST1, and pair of site-specific recombinase; and/or - a selection gene, IRES, NDST1, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS6ST1, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST1, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST3, IRES, HS6ST2, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, GLCE, IRES, HS6ST3, and a pair of site-specific recombinase; and/or - a selection gene, IRES, NDST1, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, NDST2, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS2ST1, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST1, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS3ST5, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST1, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a selection gene, IRES, HS6ST2, IRES, HS6ST3, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, GLCE, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, NDST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, HS2ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS3ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, HS3ST5, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, HS6ST1, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or - a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST3, an IRES, HS6ST2, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, GLCE, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, NDST2, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS2ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS3ST5, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST1, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- a heparan sulfate scaffold protein (such as a proteoglycan) gene, an IRES, HS6ST2, an IRES, HS6ST3, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or - SRGN, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SRGN, an IRES, HS3ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- HSPG2, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- HSPG2, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or

- HSPG2, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or

- HSPG2, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of sitespecific recombinase; and/or

- SDC1, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- SDC1, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, GLCE, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST1, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST2, an IRES, NDST2, an IRES, and a pair of site-specific recombinase; and/or

- GPC1, an IRES, HS6ST3, an IRES, NDST2, an IRES, and a pair of site-specific recombinase, optionally wherein the construct further comprises a poly A tail.

In some examples, wherein the nucleic acid construct further comprises a promoter.

In some examples, wherein the nucleic acid construct comprises a promoter for expressing the heparan sulfate scaffold protein.

In some examples, the promoter as described herein may facilitate co-expression and/or overexpression. As used herein, the terms “co-expression” and “overexpression” may be used interchangeably to refer to the expression of more than one gene in a construct.

In some examples, wherein the promoter is a chimeric promoter (ChiP).

In some examples, wherein the nucleic acid construct is a vector, optionally a landing pad vector. In another aspect, there is provided a vector comprising a nucleic acid construct as described herein.

In another aspect, there is provided a host cell and/or cell line comprising a nucleic acid construct as described herein.

In some examples, wherein the host cell and I or cell line may be a Chinese Hamster Ovary (CHO) cell line, a HEK293 cell line, or a HeLa cell line, and the like.

In some examples, the host cell and I or cell line may optionally be a CHO K1 cell line.

In some examples, the host cell and/or cell line is cultured using methods known in the art. For example, the cells may be cultured in bioreactor tubes as described herein.

Much of the metabolic engineering work performed by other studies in the art used model cell lines such as Chinese hamster ovary (CHO) or Human Embryonic Kidney (HEK293) cells. CHO cells are the dominant mammalian host cell line for producing biologies in industry. CHO cells offer many advantages over other mammalian cell lines, including the ability to easily obtain high cell densities, high protein titre, are easily cultured in chemically defined, serum-free media, and have a proven track record of producing many regulatory approved therapeutic proteins. Previous work in the art has given clear indication that the structure and composition of CHO cell heparan sulfate can be altered by the expression of heparan sulfate biosynthetic enzymes. However, the relative yield of heparan sulfate isolated using these methods is low. Factors limiting the yield of heparan sulfate include suboptimal expression of heparan sulfate biosynthetic enzymes, and a lack of GAG attachment sites (in the form of serine residues in PG core proteins), which becomes rate-limiting. Therefore, the inventors of the present disclosure hypothesize that by combining the overexpression of PG core proteins and critical heparan sulfate biosynthetic enzymes, as well as optimizing relative expression levels, the inventors of the present disclosure can alter the structure, composition and increase the overall yield of heparan sulfate produced in CHO cells.

In another aspect, there is provided a recombinant heparan sulfate produced by the host cell/cell line as described herein.

More recently, metabolic engineering has been used to alter the composition of heparan sulfate in controlled manner using CHO cells, typically to produce bioengineered heparin (HP), a highly sulphated analogue of heparan sulfate with potent anticoagulant activity. This has been achieved through the overexpression of certain heparan sulfate biosynthetic enzymes (enzymes of the NDST and 3OST family) or by overexpressing serglycin, a HSPG that is known to be decorated with heparan sulfate I heparin (HS/HP) in vivo. Other than serglycin, heparan sulfate has been reported to decorate perlecan, syndecan and glypican proteins when expressed in CHO or HEK293 cells. Overexpression of either heparan sulfate biosynthetic enzymes or HSPGs have shown success in altering the composition of heparan sulfate and increasing the amount of heparan sulfate produced, however, to date these two approaches have not yet been demonstrated to work synergistically in the same system.

Previous study in the art has given clear indication that the structure and composition of CHO cell heparan sulfate can be altered by the expression of heparan sulfate biosynthetic enzymes. However, the yield of heparan sulfate isolated using these methods is low. Factors limiting the yield of heparan sulfate include suboptimal expression of HS biosynthetic enzymes, limited availability of substrates such as uridine diphosphate (UDP)-sugars and 3’-phosphoadenosine-5’-phosphosulfate (PAPS), and a lack of glycosaminoglycan (GAG) attachment sites (in the form of serine residues in HSPG), which becomes rate-limiting.

In the present study, the inventors of the present disclosure generated transgenic CHO lines expressing one, two or three genes relating to heparan sulfate biosynthesis using the RMCE approach. The inventors of the present disclosure then proceeded to extract and analyse heparan sulfate from the conditioned media of these cell lines using several methods. After extensive screening, the inventors of the present disclosure were able to identify a recombinant heparan sulfate variant generated from transgenic CHO lines that displayed excellent potential as a media bio-additive to enhance in vitro MSC proliferation and expansion.

Without wishing to be bound by theory, it is believed that the recombinant heparan sulfate as described herein advantageously binds and stabilizes agents (such as cytokine, growth factors, morphogens, chemokines, and/or receptors) by protecting these agents from thermal and proteolytic denaturation. The recombinant heparan sulfate as described herein prolongs the half-life of agents such as cytokines, growth factors, morphogens, chemokines, and/or receptors. At the same time, the recombinant heparan sulfate as described herein advantageously sequester growth factors and bring them to the proximity of the receptors to exert its downstream function. The recombinant heparan sulfate as described herein are constructed from one or two or three or more gene constructs. As such, the recombinant heparan sulfate as described herein does not include naturally occurring heparan sulfate.

Heparan sulfate produced in the present disclosure are entirely unique and result from the insertion of a novel triple gene construct that codes for the expression of a heparan sulfate species unlike any other recombinant or naturally occurring heparan sulfate.

In another aspect, there is provided a recombinant heparan sulfate comprising a selection protein, and a heparan sulfate scaffold protein and/or a heparan sulfate modification enzyme; or a selection protein, two heparan sulfate modification enzyme; or a selection protein, one or more heparan sulfate scaffold protein, and one or more heparan sulfate modification enzyme; or a selection protein, a heparan sulfate scaffold protein, and two heparan sulfate modification enzymes; or a heparan sulfate scaffold protein and/or a heparan sulfate modification enzyme; or two heparan sulfate modification enzymes; or one or more heparan sulfate scaffold protein gene, and one or more heparan sulfate modification enzyme; or a heparan sulfate scaffold protein gene, and two heparan sulfate modification enzymes.

In some examples, wherein the protein comprises: - a selection protein, and GLCE; and/or

- a selection protein, and NDST1 ; and/or

- a selection protein, and NDST2; and/or

- a selection protein, and HS2ST1; and/or

- a selection protein, and HS3ST1 ; and/or

- a selection protein, and HS3ST5; and/or

- a selection protein, and HS6ST1 ; and/or

- a selection protein, and HS6ST2; and/or

- a selection protein, and HS6ST3; and/or

- a selection protein, and serglycin; and/or

- a selection protein, and perlecan; and/or

- a selection protein, and syndecan; and/or

- a selection protein, and glypican; and/or

- a selection protein, GLCE, and NDST2; and/or

- a selection protein, HS6ST1, and NDST2; and/or

- a selection protein, HS6ST2, and NDST2; and/or

- a selection protein, HS6ST3, and NDST2; and/or

- a selection protein, NDST1, and GLCE; and/or

- a selection protein, NDST2, and GLCE; and/or

- a selection protein, HS2ST1, and GLCE; and/or

- a selection protein, HS3ST1, and GLCE; and/or

- a selection protein, HS3ST5, and GLCE; and/or

- a selection protein, HS6ST1, and GLCE; and/or

- a selection protein, HS6ST2, and GLCE; and/or

- a selection protein, HS6ST3, and GLCE; and/or

- a selection protein, GLCE, and NDST1; and/or

- a selection protein, NDST2, and NDST1; and/or

- a selection protein, HS2ST1, and NDST1; and/or

- a selection protein, HS3ST1 , and NDST1; and/or

- a selection protein, HS3ST5, and NDST1; and/or - a selection protein, HS6ST1, and NDST1; and/or

- a selection protein, HS6ST2, and NDST1; and/or

- a selection protein, HS6ST3, and NDST1; and/or

- a selection protein, GLCE, and NDST2; and/or

- a selection protein, NDST1, and NDST2; and/or

- a selection protein, HS2ST1, and NDST2; and/or

- a selection protein, HS3ST1, and NDST2; and/or

- a selection protein, HS3ST5, and NDST2; and/or

- a selection protein, HS6ST1, and NDST2; and/or

- a selection protein, HS6ST2, and NDST2; and/or

- a selection protein, HS6ST3, and NDST2; and/or

- a selection protein, GLCE, and HS2ST1; and/or

- a selection protein, NDST1, and HS2ST1; and/or

- a selection protein, NDST2, and HS2ST1; and/or

- a selection protein, HS3ST1, and HS2ST1; and/or

- a selection protein, HS3ST5, and HS2ST1; and/or

- a selection protein, HS6ST1, and HS2ST1; and/or

- a selection protein, HS6ST2, and HS2ST1; and/or

- a selection protein, HS6ST3, and HS2ST1; and/or

- a selection protein, GLCE, and HS3ST1; and/or

- a selection protein, NDST1, and HS3ST1; and/or

- a selection protein, NDST2, and HS3ST1; and/or

- a selection protein, HS2ST1, and HS3ST1; and/or

- a selection protein, HS3ST5, and HS3ST1; and/or

- a selection protein, HS6ST1, and HS3ST1; and/or

- a selection protein, HS6ST2, and HS3ST1; and/or

- a selection protein, HS6ST3, and HS3ST1; and/or

- a selection protein, GLCE, and HS3ST5; and/or

- a selection protein, NDST1, and HS3ST5; and/or

- a selection protein, NDST2, and HS3ST5; and/or

- a selection protein, HS2ST1, and HS3ST5; and/or - a selection protein, HS3ST1, and HS3ST5; and/or

- a selection protein, HS6ST1, and HS3ST5; and/or

- a selection protein, HS6ST2, and HS3ST5; and/or

- a selection protein, HS6ST3, and HS3ST5; and/or

- a selection protein, GLCE, and HS6ST1; and/or

- a selection protein, NDST1, and HS6ST1; and/or

- a selection protein, NDST2, and HS6ST1; and/or

- a selection protein, HS2ST1, and HS6ST1; and/or

- a selection protein, HS3ST1, and HS6ST1 ; and/or

- a selection protein, HS3ST5, and HS6ST1; and/or

- a selection protein, HS6ST2, and HS6ST1; and/or

- a selection protein, HS6ST3, and HS6ST1; and/or

- a selection protein, GLCE, and HS6ST2; and/or

- a selection protein, NDST1, and HS6ST2; and/or

- a selection protein, NDST2, and HS6ST2; and/or

- a selection protein, HS2ST1, and HS6ST2; and/or

- a selection protein, HS3ST1, and HS6ST2; and/or

- a selection protein, HS3ST5, and HS6ST2; and/or

- a selection protein, HS6ST1, and HS6ST2; and/or

- a selection protein, HS6ST3, and HS6ST2; and/or

- a selection protein, GLCE, and HS6ST3; and/or

- a selection protein, NDST1, and HS6ST3; and/or

- a selection protein, NDST2, and HS6ST3; and/or

- a selection protein, HS2ST1, and HS6ST3; and/or

- a selection protein, HS3ST1, and HS6ST3; and/or

- a selection protein, HS3ST5, and HS6ST3; and/or

- a selection protein, HS6ST1, and HS6ST3; and/or

- a selection protein, HS6ST2, and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1 , and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and GLCE; and/or - a HS scaffold protein (such as a proteoglycan), HS3ST1 , and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and GLCE; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1 , and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and NDST1; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1, and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1 , and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and NDST2 and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and NDST2; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1 , and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and HS2ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS3ST1; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1 , and HS3ST1 ; and/or - a HS scaffold protein (such as a proteoglycan), NDST2, and HS3ST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1, and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1 , and HS3ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1, and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and HS3ST5; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS6ST1; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1 , and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1 , and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and HS6ST1; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and HS6ST1 ; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), NDST1, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1 , and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1 , and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST3, and HS6ST2; and/or

- a HS scaffold protein (such as a proteoglycan), GLCE, and HS6ST3; and/or - a HS scaffold protein (such as a proteoglycan), NDST1 , and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), NDST2, and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), HS2ST1, and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST1 , and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), HS3ST5, and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST1, and HS6ST3; and/or

- a HS scaffold protein (such as a proteoglycan), HS6ST2, and HS6ST3; and/or

- serglycin, GLCE, and NDST2; and/or

- serglycin, HS6ST1, and NDST2; and/or

- serglycin, HS6ST2, and NDST2; and/or

- serglycin, HS6ST3, and NDST2; and/or

- perlecan, GLCE, and NDST2; and/or

- perlecan, HS6ST1, and NDST2; and/or

- perlecan, HS6ST2, and NDST2; and/or

- perlecan, HS6ST3, and NDST2; and/or

- syndecan, GLCE, and NDST2; and/or

- syndecan, HS6ST1, and NDST2; and/or

- syndecan, HS6ST2, and NDST2; and/or

- syndecan, HS6ST3, and NDST2; and/or

- glypican, GLCE, and NDST2; and/or

- glypican, HS6ST1, and NDST2; and/or

- glypican, HS6ST2, and NDST2; and/or

- glypican, HS6ST3, and NDST2.

In some examples, wherein the selection protein is an antibiotics resistant protein, such as, but is not limited to, puromycin resistant, neomycin resistant (NeoR), blasticidin resistant (BsdR), hygromycin resistant (HygR), bleomycin resistant (BleoR), and the like.

In some examples, the selection proteins may confer resistance to selective antibiotics G418/geneticin, blasticidin, hygromycin B and zeocin, respectively. In some examples, wherein the heparan sulfate scaffold protein is a mammalian heparan sulfate proteoglycan.

In some examples, heparan sulfate proteoglycan may be a human heparan sulfate proteoglycan, a non-human primates heparan sulfate proteoglycan, a pig heparan sulfate proteoglycan, a mouse heparan sulfate proteoglycan, a rat heparan sulfate proteoglycan, and the like. In some examples, the heparan sulfate proteoglycan may include, but is not limited to serglycin, perlecan, syndecan, glypican, syndecan 2, syndecan 3, syndecan 4, glypican 2, glypican 3, glypican 4, glypican 5, glypican 6, and the like.

In another aspect, there is provided a recombinant heparan sulfate for use in enhancing cell proliferation.

In some examples, the cell is a stem cell. In some examples, the cell is a human mesenchymal stem cell.

In another aspect, there is provided a recombinant heparan sulfate for use in enhancing stability of a component such as, but is not limited to, cytokine, growth factors, morphogens, chemokines, receptors, and the like.

In some examples, the cytokine may be an interleukin. In some examples, the cytokine may be one or more of interleukin 1 (IL1), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL12, tumor necrosis factor a (TN Fa), interferon y (IFNy), and the like. In some examples, the cytokine is IL2. In some examples, the chemokines may include, but is not limited to stromal cell-derived factor 1 a (SDF1a) (C-X-C motif chemokine ligand 12 - CXCL12), IL10, I L14, and the like.

In some examples, the growth factors may include, but is not limited to, fibroblast growth factor 1 (FGF1), FGF2, FGF4, FGF6, FGF7, FGF8, FGF9, FGF10, FGF16, FGF17, FGF20, bone morphogenetic protein 2 (BMP2), BMP4, BMP6, BMP7, vascular endothelial growth factor (VEGF), transforming growth factor pi (TGFpi), sonic hedgehog (SHH), platelet-derived growth factor-AB (PDGF-AB), PDGF-BB, heparin- binding hepatocyte growth factor (HB-GFH), and the like.

In some examples, the receptors may include, but is not limited to vascular endothelial growth factor receptor II (VEGFRII), bone morphogenetic protein receptor II (BMPRII), fibroblast growth factor receptor 1 (FGFR1), C-X-C chemokine receptor type 4 (CXCR4), and the like.

The inventors of the present disclosure compared the heparan sulfate produced in these different CHO expressing cell lines with regards to the heparan sulfate yield, FGF2 binding affinity and hMSC stimulating capabilities. The inventors of the present disclosure found that overexpressing the heparan sulfate scaffold proteins in CHO cells increases the yield of heparan sulfate produced, with serglycin producing the highest yield among the four scaffold proteins tested. FiG. 12 shows the characterisation of HS- TG. When serglycin was co-expressed with dual heparan sulfate modification enzyme genes, the heparan sulfate produced with the triple gene construct Serglycin-Hs6st3- NDST2 (termed HS-TG) had the best yield, most favourable composition, highest binding affinity and enhanced MSC proliferation (FIG. 12).

In addition, the inventors of the present disclosure further screened a panel of triple gene heparan sulfate variants in an interleukin-2 stability assay. Here, each heparan sulfate variant (1 pg) was incubated with interleukin-2 (IL2) (20 pg) for 6 hours at 37°C, then subsequently used to stimulate an IL2 reporter cell line, HEK-Blue-IL2. The HEK-Blue-IL2 line has been engineered to express all relevant signalling components of the IL2 pathway and IL2 promoter-driven alkaline phosphatase, which is used to measure IL2 activity. All triple gene heparan sulfate variants can enhance IL2 stability, however the Serglycin-GLCE-NDST2 day 14 variant gave the greatest increase in stability (FIG. 13).

The inventors of the present disclosure designed combinations of heparan sulfate modification enzymes with scaffold proteins to optimise the production of specific heparan sulfate variants in CHO cells that can bind with high affinity to FGF2 and can stimulate hMSC proliferation in culture. In addition, the inventors of the present disclosure compared the different heparan sulfate variants produced and found that the best performing heparan sulfate was produced by the gene combination of Serglycin- Hs6st3-NDST2. The production of the desirable heparan sulfate depends on the expression of three genes at specific levels. Precise control of multiple gene expression can be achieved by optimization of regulatory elements in the plasmid vectors and sitespecific integration of plasmid vectors into the predetermined active genomic sites in mammalian cells. The usage of the technology can be traced by the unique disaccharide profile described in the present disclosure.

In another aspect, there is provided a method of culturing a cell and/or inducing a cell proliferation and/or expanding a cell, the method comprising culturing the cell in a media comprising the recombinant heparan sulfate as described herein.

In some examples, the cell is a stem cell. In some examples, the stem cell is a human mesenchymal stem cell. In some examples, wherein the method is an in vitro method.

In some examples, the media further comprising a cytokine, a growth factor, a morphogen, a chemokine, and the like.

In some examples, the media further comprising a cytokine, a growth factor, a morphogen, a chemokine, and the like.

In some examples, the cytokine is an interleukin. In some examples, the cytokine is IL1 , IL2, IL3, IL4, IL5, IL6, and the like. In some examples, the cytokine is IL2.

In some examples, the growth factors may include, but is not limited to, FGF2, BMP2, VEGF, TGF 1 , and the like.

In another aspect, there is provided a method of generating a recombinant heparan sulfate comprising transfection of the vector as described herein into a cell line.

In some examples, wherein said cell line is a master cell line generated by transfection (such as nucleofection) of a landing pad vector into the cell line.

In some examples, wherein the landing pad vector comprises a selection gene, a promoter, and a site-specific recombinase placed at both ends of the landing pad vector.

In some examples, the landing pad vector may be the HYG expression cassette as described herein. In some examples, the landing pad vector may comprise a hygromycin resistant gene (HYG), a chimeric promoter (ChiP). In some examples, the chimeric promoter may comprise/consist of CMV enhancer, hCMV core promoter and hCMV intron A.

In some examples, the site integration of the landing pad vector is tested to ensure optimal integration into an area of the genome that produces stable and high producing cells.

In some examples, wherein the nucleic acid construct as described herein is transfected into the master cell line in the presence of a vector expressing a recombinase, optionally the recombinase is different from the heterologous recombinase used in the nucleic acid construct.

In some examples, the master cell line is transfected with the nucleic acid construct as described herein via recombinase-mediated-cassette-exchange (RMCE). In some examples, the recombinase may be FLPe, FRT3, FRT, Cre-LoxP, BXB1-attP- attB, and the like.

In some examples, wherein the method further comprising screening the cell lines for integration by southern blotting. In some examples, the integration is a single copy and/or site-specific integration. In some examples, the method further comprises the step of selecting a master cell bearing a single landing pad vector.

In some examples, wherein the heparan sulfate is a heparan sulfate variant and/or a heparan sulfate scaffold protein and/or heparan sulfate modification enzyme and/or combination thereof.

In some examples, wherein the heparan sulfate modification enzyme may include, but is not limited to, N-Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, NDST3, NDST4, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-0- Sulfotransferase 1 (HS2ST7), HS3ST1, HS3ST5, HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS6ST3, and the like, and combination and/or isoforms thereof.

In some examples, wherein the heparan sulfate scaffold protein may be a mammalian heparan sulfate scaffold protein.

In some examples, heparan sulfate proteoglycan may be a human heparan sulfate proteoglycan, a non-human primates heparan sulfate proteoglycan, a pig heparan sulfate proteoglycan, a mouse heparan sulfate proteoglycan, a rat heparan sulfate proteoglycan, and the like. In some examples, the heparan sulfate proteoglycan may include, but is not limited to serglycin, perlecan, syndecan, glypican, syndecan 2, syndecan 3, syndecan 4, glypican 2, glypican 3, glypican 4, glypican 5, glypican 6, and the like, and combination thereof.

In some examples, wherein the method further comprises culturing the cell line in a media suitable for heparan sulfate production.

In some examples, wherein the method further comprises isolating heparan sulfate from the media comprising the cell line.

In some examples, the heparan sulfate may be isolated from the media of the cell lines by methods known in the art. In some examples, the heparan sulfate may be isolated by methods as described herein.

In some examples, wherein the method further comprises analyzing the purified heparan sulfate for disaccharide composition.

In some examples, the heparan sulfate may be analysed for disaccharide composition by methods known in the art. In some examples, the heparan sulfate may be analyzed for disaccharide composition by methods as described herein.

The inventors of the present disclosure have generated CHO cells expressing different heparan sulfate (HS) variants by transfecting the following constructs into CHO cells (FIG.6). A total of nine different heparan sulfate modification enzymes and four heparan sulfate scaffold proteins are used in the present disclosure. The nine heparan sulfate modification enzymes are N-Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-O-Sulfotransferase 1 (Hs2st1), Hs3st1 , Hs3st5, Hs6st1 , Hs6st2 and Hs6st3. The four heparan sulfate scaffold proteins are serglycin (SRGN), glypican (GPC1), perlecan domain 1 (HSPG2 and syndecan 1 (SDC1).

The inventors of the present disclosure have invented a method of producing highly potent heparan sulfate that can bind with high affinity to FGF-2 by combining expression of specific heparan sulfate modification enzymes in CHO cells. This heparan sulfate variant termed CHO-TG (i.e. , serglycin-6OST3-NDST2 construct) can enhance human mesenchymal stem cell proliferation in vitro to a greater extent than the addition of FGF-2 alone. In addition to heparan sulfate-TG, the inventors of the present disclosure have generated four triple gene variants, all of which can improve the stability of IL-2.

Using a metabolic engineering approach, the inventors of the present disclosure have transfected CHO cells with a number of constructs containing different enzymes involved in the modification of heparan sulfate (HS). In the present disclosure, the inventors of the present disclosure generated transgenic CHO lines expressing one, two or three genes relating to heparan sulfate biosynthesis. The inventors of the present disclosure then proceeded to extract and analyse heparan sulfate from the conditioned media of these cell lines using several methods. The heparan sulfate can then be isolated from the conditioned culture media, then assessed using multiple screening methods to determine composition, bioactivity, and relative affinity toward various proteins of interest. Using data from heparan sulfate variants generated from cell lines expressing individual heparan sulfate enzymes, the inventors of the present disclosure have been able to further engineer CHO lines to express combinations of multiple heparan sulfate enzymes. This has allowed the inventors of the present disclosure to tune the composition and total yield of heparan sulfate produced by each cell line, which facilitates the production of customisable heparan sulfate variants tuned to bind and enhance the bioactivity of specific heparin-binding proteins and growth factors, including FGF-2 and IL2. After extensive screening, the inventors of the present disclosure were able to identify a recombinant heparan sulfate variant generated from transgenic CHO lines that displayed excellent potential as a media bio-additive to enhance MSC proliferation and expansion. In another aspect, there is provided a kit for generating a recombinant heparan sulfate comprising a nucleic acid construct as described herein, and an instruction to perform the method as described herein.

In some examples, wherein the recombinant heparan sulfate comprises a heparan sulfate scaffold protein and/or heparan sulfate modification enzyme.

In some examples, wherein the heparan sulfate modification enzyme may include, but is not limited to, N-Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, NDST3, NDST4, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-0- Sulfotransferase 1 (HS2ST7), HS3ST1, HS3ST5, HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS6ST3, and combination and/or isoforms thereof.

In some examples, wherein the heparan sulfate scaffold protein is a mammalian heparan sulfate scaffold protein.

In some examples, heparan sulfate proteoglycan may be a human heparan sulfate proteoglycan, a non-human primates heparan sulfate proteoglycan, a pig heparan sulfate proteoglycan, a mouse heparan sulfate proteoglycan, a rat heparan sulfate proteoglycan, and the like. In some examples, the heparan sulfate proteoglycan may include, but is not limited to serglycin, perlecan, syndecan, glypican, syndecan 2, syndecan 3, syndecan 4, glypican 2, glypican 3, glypican 4, glypican 5, glypican 6, and the like, and combination thereof.

In another aspect, there is provided a kit for culturing a cell (such as a stem cell, optionally a human mesenchymal stem cell) comprising a recombinant heparan sulfate as described herein, and an instruction to perform the method of culturing a cell as described herein.

In some examples, wherein the heparan sulfate comprises a heparan sulfate scaffold protein and/or a heparan sulfate modification enzyme.

In some examples, wherein the heparan sulfate modification enzyme may include, but is not limited to, N-Deacetylase And N-Sulfotransferase 1 (NDST1), NDST2, NDST3, NDST4, Glucuronic Acid Epimerase (GLCE), Heparan Sulfate 2-0- Sulfotransferase 1 (HS2ST7), HS3ST1, HS3ST5, HS3ST1, HS3ST2, HS3ST3A, HS3ST3B, HS3ST4, HS3ST5, HS3ST6, HS6ST1, HS6ST2, HS6ST3, and combination and/or isoforms thereof.

In some examples, wherein the heparan sulfate scaffold protein is a mammalian heparan sulfate scaffold protein. In some examples, heparan sulfate proteoglycan may be a human heparan sulfate proteoglycan, a non-human primates heparan sulfate proteoglycan, a pig heparan sulfate proteoglycan, a mouse heparan sulfate proteoglycan, a rat heparan sulfate proteoglycan, and the like. In some examples, the heparan sulfate proteoglycan may include, but is not limited to serglycin, perlecan, syndecan, glypican, syndecan 2, syndecan 3, syndecan 4, glypican 2, glypican 3, glypican 4, glypican 5, glypican 6, and the like, and combination thereof.

Cell culture and media for maintenance of CH O K1 master cell line (MCL)

In some examples, the method comprises generating a cell line (such as CHO K1 master cell line) by transfection (such as nucleofection) of a vector (such as a landing pad vector) into cells (such as CHO K1 cells), followed by screening clones for integration (such as single copy integration) by methods known in the art (such as southern blotting). In some examples, the vector (such as landing pad vector) expresses a gene (such as hygromycin resistant gene) using a promoter (such as chimeric promoter) which consisted of an enhancer (such as murine CMV M11788 enhancer), a core promoter (such as hCMV) and an intron (such as hCMV intron A: M60321). In some examples, the expression cassette (such as HYG expression cassette) was flanked by genes (such as FRT3 and FRT). In some examples, an impaired gene (such as puromycin resistant gene lacking start codon ((ATG-)Puro) followed by a signal (such as SV40 polyadenylation signal (SpA)) was placed downstream of a gene (such as FRT) for selecting correct cassette exchange by recombination (such as recombinase- mediated cassette exchange). In some examples, the cell line (such as MCL) was confirmed to contain one copy of the vector (such as landing pad vector) at an integration site (such as a single integration site) by methods known in the art (such as southern blotting and targeted locus amplification (TLA) analysis). In some examples, the cell line (such as MCL) was grown in a medium (such as protein free medium (PFM) with 50% HyQ PF (GE Healthcare Life Sciences) and 50% CD CHO (Thermo Fisher Scientific)) supplemented with salt (such as 1g/L sodium carbonate), amino acid (such as 6mM glutamine), and surfactant (such as 0.1% Pluronic F-68) in a shaker (such as a humidified Kuhner shaker) with CO2 (such as 8% CO2) at 37°C. In some examples, routine subculture was conducted (such as every 3 to 4 days) by seeding cells (such as at density of 3x10 ⁵ cells/mL in 15 mL of fresh medium) in shake flasks. In some examples, cell density and viability were determined by methods known in the art (such as trypan blue exclusion method on Vi-Cell XR viability analysers). Construction of targeting vectors forexpression of heparan sulfate modification enzymes

In some examples, the method comprises synthesizing the target vectors containing a modification gene (such as single heparan sulfate) or scaffold by an external company (such as Genscript). In some examples, the method comprises generating modified gene constructs (such as dual heparan sulfate modification) with insertion sites in the vector backbone by an external company (such as Genscript). In some examples, the vector backbone may include one, or two, or three, or four, or five, or six insertion sites. In some examples, the vector backbone includes two insertion sites. In some examples, the restriction enzyme sites (such as Mlul and Xhol for position 1 ; Mfel and EcoRI for position 2) were used to replace the gene in position 1 and position 2.

In some examples, the method comprises generating modified gene constructs (such as triple heparan sulfate modification) where the antibiotic (such as puromycin) in the gene construct (such as dual gene construct) were switched out with another protein (such as serglycin) using restriction enzyme sites (such as EcorV and Notl). In some examples, a part of the construct (such as the pA tail) was removed and replaced with another part (such as wild type IRES) using restriction enzyme sites (such as EcoRI and AsiSI).

Generating stable heparan su ate-producing cell lines via recombinase-mediated- cassette-exchange (RMCE)

In some examples, the method comprises co-transfecting a cell line (such as the MCL) with a first vector (such as an appropriate targeting vector) and a second vector (such as a vector expressing a recombinase (such as FLPe). In some examples, the cotransfecting may be performed using a commercially available kit (such as Amaxa SG Cell Line 4D-Nucleofector ® X Kit Land program FF-137 (Lonza)). In some examples, cells were transfected with a plasmid vector and recombinase plasmid vector in circular format. In some examples, the transfected cells were resuspended in medium (such as protein free medium (PFM)) preloaded in culture plates and incubated in incubators.

In some examples, the transfected cells were collected by centrifugation and resuspended in medium (such as PFM) in flasks (such as shake flasks) in a shaker (such as humidified Kuhner shaker) with CO2 (such as 8% CO2) at 37°C post transfection (such as 6 h, 12 h, 18 h, 24 h, 36 h, 48 h or 72 h post transfection). In some examples, the transfected cells were collected by centrifugation at 24 h post transfection. In some examples, the method comprises subjecting the transfected cells to selection in medium (such as PFM) containing antibiotics (such as about 1 pg/mL, 5 pg/mL, 10 pg/mL, 15 pg/mL, 20 pg/mL, 25 pg/mL, 30 pg/mL, 40 pg/mL, or 50 pg/mL puromycin), such as one day, two days, three days, four days, five days, six days later. In some examples, the transfected cells were subjected to selection in PFM containing 20 pg/mL puromycin four days later.

In some examples, selection was continued for such as 3 days, 4 days, 5 days, 6 days, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks by passaging in selection medium every 3 to 4 days. In some examples, selection was continued for 2 weeks by passaging in selection medium every 3 to 4 days.

In some examples, the stably transfected cell pools were deemed established when cell viabilities recovered over 80%, 85%, 90%, or 95%. In some examples, the stably transfected pools were deemed established when cell viabilities recovered over 95%.

Genomic integration analysis

In some examples, the method comprises checking for correct cassette recombination by extracting DNA (such as genomic DNA I gDNA) from cells using a commercially available kit. In some examples, the DNA was used as a template for PCR using a mixture. In some examples, primer sets flanking the integration sites (such as 5’ and 3’ integration sites) were used to check for successful integration of the transgene. In some examples, the primers used in the PCR (such as junction PCR) are listed in Table 2. In some examples, the PCR products were visualized on agarose gel stained with a fluorescent dye.

Fed-batch culture for heparan sulfate production

In some examples, the method comprises generating heparan sulfate by seeding the recovered cell lines in tubes containing medium (such as PFM). In some examples, medium with sugar) was added to each tube spin on day, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and / or 15. In some examples, medium with sugar was added to each tube spin on day 3, 5, 7, 9 and 11. In some examples, the cultures were performed more than once.

In some examples, the method comprises maintaining sufficient sugar level by supplementing sugar when the sugar concentration in the culture medium drops below about 0.5 g/L, 1 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 5.5 g/L, or 6 g/L. In some examples, the method comprises maintaining sufficient glucose level by supplementing 3 g/L of glucose when glucose concentration in the culture medium drops below 3 g/L.

In some examples, heparan sulfate in the culture medium was harvested on day

I , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 by centrifugation (and collecting the supernatant. In some examples, the heparan sulfate in the culture medium was harvested on day 7 and day 14 by centrifugation and collecting the supernatant.

Single cell cloning of transfected cells

In some examples, cell clones (such as CHO cell clones) were isolated for heparan sulfate composition analysis and yield. In some examples, transfected cells (such as CHO cells) were diluted to a final concentration of about 100 cells I mL, 200 cells I mL, 300 cells I mL, 400 cells I mL, 500 cells I mL, 600 cells I mL, 700 cells I mL, 800 cells /mL, 900 cells I mL or 1000 cells I mL in commercially available media. In some examples, transfected cells were diluted to a final concentration of about 600 cells I mL.

In some examples, about 1 ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml, 10ml, 11 ml, 12ml, 13ml, 14ml, 15ml, 20ml cell suspension were aliquoted into each cell culture dish and incubated with CO2 (such as 8% CO2) at 37°C for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,

I I , 12, 13, 14, 15, 16, 17, 18, 19, 20 days. In some examples 12mL of cell suspension were aliquoted into each petri dish and incubcated with 8% CO2 at 37°C for 14 days.

In some examples, cell colonies were picked up and transferred into cell culture plates and allowed to grow till about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% confluent. In some examples, cell colonies were picked up and transferred into 24 well plates and allowed to grow till about 50% confluent.

In some examples, cells were transferred into cell culture tubes containing about 1 mL, 2mL, 3mL, 4mL, 5mL, 6mL, 7mL, 8mL, 9mL, 10mL of protein-free media. In some examples, cells were transferred into cell culture tubes containing about 5 mL of protein- free media.

Isolation of heparan sulfate from conditioned media

In some examples, the method comprises filtering the medium through a membrane treated with a lyase, hydrolase and endonucleases in the presence of an inorganic compound for about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 , 22 h, 23 h, 24 h, 36 h, or 48 h at 37°C. In some examples, the CHO condition media was vacuum filtered through a 0.2 pm membrane treated with about 5, 10, 20, 30, 40, 50, 100 or 1000 mill chondroitinase ABC (Merck); about 1 , 5, 10, 20, 40, 50 or 100 ml U neuraminidase (Merck); about 100, 200, 300, 400, 500, 1000, or 2000 kunitz units DNAse I (Merck); and about 50, 100, 200, 300, 400, 500 or 1000 units RNase IA (Merck) in the presence of 0.02% (w/v) sodium azide for about 4, 6, 8, 16, 24, 48 or 72 h at 37°C. In some examples, the CHO conditioned media was vacuum filtered through a 0.2 pm membrane treated with 10 mIU chondroitinase ABC (Merck), 10 mIU neuraminidase (Merck), 1000 kunitz units DNAse I (Merck) and 500 units RNAse IA (Merck) in the presence of 0.02% (w/v) sodium azide for about 16 h at 37°C.

In some examples, the method comprises adding a protease (such as pronase, from streptomyces griseus) to a final concentration such as about 50 pg/m, 100 pg/mL, 150 pg/mL, 200 pg/mL, 250 pg/mL, 300 pg/mL, 350 pg/mL, 400 pg/mL, 450 pg/mL, 500 pg/mL, 550 pg/mL, 600 pg/mL, 700 pg/mL, 800 pg/mL, 900 pg/mL, or 1000 pg/mL; and media incubated for a further such as about 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 16 h, 24 h, 36 h, 48 h at 37°C. In some examples, pronase (from streptomyces griseus) was added to a final concentration of 500 pg/mL and media incubated for a further 4 h at 37°C.

In some examples, the method comprises cooling the media to room temperature and isolating the heparan sulfate using a column attached to a chromatography system. In some examples, the column may include an anion exchange resin, with functional groups such as but is not limited to DEAE, Capto Q, Q-XL or Q ImpRes, and the like. In some examples, about 15 mL, 20 mL, 25 mL, 50 mL, 100 mL, 250 mL, 500 mL, 1000 mL, 1250 mL, 1500 mL, or 2000 mL media was applied to the column. In some examples, the column volume (C.V.) is about 1 mL, 5 mL, 10 mL, 20 mL, 50 mL, 100 mL, 200 mL, or 500 mL. In some examples, the media was applied to the column and washed with column volumes of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, or 300 mM sodium chloride; column volumes of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM sodium phosphate monobasic; column volumes of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM sodium phosphate dibasic, in pH such as pH 7.2, followed by column volumes of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, or 300 mM sodium chloride., In some examples, 25 mL of media was applied to the column, then washed with 10 C.V. of 150 mM sodium chloride, 20 mM sodium phosphate monobasic, 20 mM sodium phosphate dibasic, pH 7.2, followed by 10 C.V. 200 mM sodium chloride.

In some examples, heparan sulfate was eluted in water and subsequently desalted using a desalting column attached to a chromatography system In some examples, the desalted heparan sulfate isolates were frozen and lyophilised to dryness prior to weighing.

Disaccharide compositional analysis

In some examples, the method comprises treating the purified heparan sulfate samples of purified heparan sulfate samples with lyase (such as about 0.1 mill, 0.5 mIU, 1 mIU, 1.5 mIUm 2 mIU, 2.5 mIU, 3 mIU, or 5 mIU of heparinase I, II and III) in digest buffer for about 3 h, 6 h, 12 h, 18 h, 24 h, 36 h, or 48 h at 37°C. In some examples, the digest buffer comprises about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, or 300 mM sodium acetate; and about 0.1 mM, 0.5 mM, 1 mM, 1 .5 mM, 2 mM, 3 mM, 4 mM, or 5 mM calcium acetate, at pH 7. In some examples, the method comprises treating 500 pg purified heparan sulfate samples with 2 mIU of heparinase I, II and III in digest buffer (100 mM sodium acetate, 1 mM calcium acetate, pH 7) for 24 h at 37°C.

In some examples, a second aliquot of lyase (such as about 0.1 mIU, 0.5 mIU, 1 mIU, 1.5 mIUm 2 mIU, 2.5 mIU, 3 mIU, or 5 mIU of heparinase I, II and III) was added to each sample after about 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 60 h, or 72 h; and the digest allowed to proceed for a further such as 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 60 h, or 72 h at 37°C. In some examples, after a second 2 mIU aliquot of heparinase I, II and III were added after 24 h to each sample and the digest allowed to proceed for a further 24 h at 37°C.

In some examples, once complete, the samples were heated at about 70°C, 80°C, 90°C, 95°C, or 100°C for about 1 min, 2 min, 3 min, 4 min, 5 min, 8 min, 10 min, 15 min, or 20 min; cooled and disaccharides isolated via chromatography using a column under flow (such as about 0.1 ml /min, 0.5 ml/min, 0.75 ml/min, 1 ml/ min, 1.5 ml/ min, 1.75 ml/ min, 2 ml / min, or 5 ml / min isocratic flow) in salt buffer (such as about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250 mM, or 300 mM ammonium carbonate). In some examples, once complete, samples were heated at 100°C for 5 min, cooled and disaccharides isolated via size exclusion chromatography using an Akta Pure 25M with Superdex Peptide 10/300 gl SEC column under isocratic flow (0.75 ml/min) in 200 mM ammonium bicarbonate.

In some examples, the method comprises freezing and lyophilising the isolated twice to remove the salt buffer. In some examples, the disaccharides were resuspended in water, filtered and applied to a column. In some examples, the column may include a ProPac PA-1 anion exchange column.

In some examples, the disaccharides were eluted using a salt gradient at about 0.1mL/min, 0.5mL/min, 0.75mL/min, 1mL/min, 1.5mL/min, or 2mL/min (40°C column temperature). In some examples, buffer B may consist of sodium chloride. In some examples, the disaccharides were eluted using a salt gradient (buffer A = water, pH 3.5, buffer B = 2M sodium chloride, pH 3.5) at 1mL /min (40°C column temperature) using the following gradient: 0% B for 1 min, 0 to 35% B for 31 min, 35 to 65% B for 15 min, 100% B for 10 min, 0% B for 3 min.

In some examples, the eluted disaccharides were monitored at a fluorescent wavelength and identified based on elution points of known disaccharide standards. In some examples, the percentage composition was calculated based on the division of the peak area of each disaccharide by the sum of all peak areas, multiplied by 100. In some examples, the data represents mean ± S.D. of the replicate analysis of independent digests .

MSC proliferation

In some examples, the method comprises maintaining the cells in media in 5% CO2 at 37°C and used at a passage for proliferation assays. In some examples, the maintenance media may include medium, serum, amino acid, and antibiotics. In some examples, the maintenance media to maintain human MSCs include DM EM (low glucose), 10% [v/v] foetal calf serum, 4 mM L-glutamine, 100 lll/mL penicillin and 100 pg/mL streptomycin.

In some examples, cells were seeded in a culture plastic plate and allowed to attach prior to treatment with growth factor or heparan sulfate variants in maintenance media. In some examples, the treatment may include about 0.5 ng/mL, 1 ng/mL, 1.25 ng/mL, 1.5 ng/mL, 2 ng/mL, 3 ng/mL, or 5 ng/mL of FGF-2. In some examples, the treatment may include about 5 pg/mL, 10 pg/mL, 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 80 pg/mL, or 100 pg/mL of heparan sulfate variants. In some examples, cells were seeded in a culture plastic plate and allowed to attach for 24 h prior to treatment with 1.25 ng/mL FGF-2 or 50 pg/mL heparan sulfate variants in maintenance media.

In some examples, growth factor ± heparan sulfate were incubated at room temperature (such as for 5 min, 10 min, 15 min, 20 min, 25 min, or 30 min) in buffer solution prior to adding to the cell media. In some examples, growth factor ± heparan sulfate were incubated at room temperature for 10 min in 20 pL PBS prior to adding to the cell media.

In some examples, an inhibitor in DMSO was combined with cytokines or buffer at concentrations of about 5 nM, 10 nM, 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM, 250 nM, or 300 nM. In some examples, PD173074 in DMSO was combined with heparan sulfate, FGF2, PBS at concentrations of 25 nM, 50 nM or 100 nM.

In some examples, a blocking antibody was added at dilutions of about 1 :100, 1 :250, 1 :500, 1 :1000, 1 :2000, 1 :3000, 1 :5000 or 1 :10000 to treatments prior use. In some examples, FGFR1 blocking antibody IMBR1 was added at 1 :1000 or 1 :2000 to treatments prior to use.

In some examples, cells were cultured for about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 ,

12, 13, or 14 days (with a second treatment at about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,

13, or 14 days) and harvested at about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days for analysis using an instrument. In some examples, cells were cultured for 8 days (with a second treatment at day 3) and harvested at day 8 for viability analysis using a Guava EasyCyt instrument.

The term "associated with", used herein when referring to two elements refers to a broad relationship between the two elements. The relationship includes, but is not limited to a physical, a chemical or a biological relationship. For example, when element A is associated with element B, elements A and B may be directly or indirectly attached to each other, or element A may contain element B or vice versa.

The term "and/or", e.g., "X and/or Y" is understood to mean either "X and Y" or "X or Y" and should be taken to provide explicit support for both meanings or for either meaning. Further, in the description herein, the word “substantially” whenever used is understood to include, but not restricted to, "entirely" or “completely” and the like. In addition, terms such as "comprising", "comprise", and the like whenever used, are intended to be non-restricting descriptive language in that they broadly include elements/components recited after such terms, in addition to other components not explicitly recited. For example, when “comprising” is used, reference to a “one” feature is also intended to be a reference to “at least one” of that feature. Terms such as “consisting”, “consist”, and the like, may in the appropriate context, be considered as a subset of terms such as "comprising", "comprise", and the like. Therefore, in embodiments disclosed herein using the terms such as "comprising", "comprise", and the like, it will be appreciated that these embodiments provide teaching for corresponding embodiments using terms such as “consisting”, “consist”, and the like. Further, terms such as "about", "approximately" and the like whenever used, typically means a reasonable variation, for example a variation of +/- 5% of the disclosed value, or a variance of 4% of the disclosed value, or a variance of 3% of the disclosed value, a variance of 2% of the disclosed value or a variance of 1 % of the disclosed value.

Furthermore, in the description herein, certain values may be disclosed in a range. The values showing the end points of a range are intended to illustrate a preferred range. Whenever a range has been described, it is intended that the range covers and teaches all possible sub-ranges as well as individual numerical values within that range. That is, the end points of a range should not be interpreted as inflexible limitations. For example, a description of a range of 1% to 5% is intended to have specifically disclosed sub-ranges 1% to 2%, 1% to 3%, 1 % to 4%, 2% to 3% etc., as well as individually, values within that range such as 1%, 2%, 3%, 4% and 5%. It is to be appreciated that the individual numerical values within the range also include integers, fractions and decimals. Furthermore, whenever a range has been described, it is also intended that the range covers and teaches values of up to 2 additional decimal places or significant figures (where appropriate) from the shown numerical end points. For example, a description of a range of 1 % to 5% is intended to have specifically disclosed the ranges 1.00% to 5.00% and also 1.0% to 5.0% and all their intermediate values (such as 1.01%, 1.02% ... 4.98%, 4.99%, 5.00% and 1.1 %, 1.2% ... 4.8%, 4.9%, 5.0% etc.,) spanning the ranges. The intention of the above specific disclosure is applicable to any depth/breadth of a range.

Additionally, when describing some embodiments, the disclosure may have disclosed a method and/or process as a particular sequence of steps. However, unless otherwise required, it will be appreciated that the method or process should not be limited to the particular sequence of steps disclosed. Other sequences of steps may be possible. The particular order of the steps disclosed herein should not be construed as undue limitations. Unless otherwise required, a method and/or process disclosed herein should not be limited to the steps being carried out in the order written. The sequence of steps may be varied and still remain within the scope of the disclosure.

Furthermore, it will be appreciated that while the present disclosure provides embodiments having one or more of the features/characteristics discussed herein, one or more of these features/characteristics may also be disclaimed in other alternative embodiments and the present disclosure provides support for such disclaimers and these associated alternative embodiments.

EXPERIMENTAL SECTION

MATERIAL & METHODS

Materials

All chemicals and reagents were purchased from Merck unless otherwise stated. HiTrap Capto Q 5 mL columns, HiPrep 26/10 desalting columns and series S (SA) sensor chips were purchased from Cytiva (U.S.A.). Recombinant FGF2, FGFRIa lllb Fc chimera, FGFR2a lllb Fc chimera, VEGF s, BMP-2, PDGF-AB, PDGF-BB and SDF1a proteins, Mouse anti-Human FGFR1 (Monoclonal Mouse lgG2B, R&D systems cat. # MAB658), Mouse anti-Human FGFR2 (Monoclonal Mouse IgGi, R&D systems cat. # MAB684) and Goat anti-Human FGF2 (Polyclonal Goat IgG, R&D systems cat. # AB- 233-NA) were purchased from R&D systems. The IMBR1 FGFR1 blocking antibody was produced as previously described by a study in the art (Ling et al, Molecular Cancer 2015 Vol. 14 Issue 1 Pages 136) . Heparinase I, II and III, and unsaturated heparan sulfate disaccharides were purchased from Iduron (U.K.). Kinetichrome anti-XAE kits were purchased from Provision Kinetics (U.S.A.). ProPac PA1 (50 x 4 mm and 250 x 4 mm) columns were purchased from Thermo Fisher Scientific (U.S.A.).

Cell culture and media for maintenance of CHO K1 master cell line (MCL)

The CHO K1 master cell line (MCL) was generated by transfection of a landing pad vector into CHO K1 cells (ATCC), followed by Southern blot screening of clones for single copy integration. The landing pad vector drives a hygromycin resistant gene (HYG) and a Zeocin resistant gene (Zeo) with a chimeric promoter (ChiP) which consisted of the murine CMV enhancer, the hCMV core promoter and the hCMV intron A. The HYG expression cassette was flanked by FRT3 and FRT sites. An impaired puromycin resistant gene lacking a start codon ((ATG-)Puro) followed by the simian virus 40 (SV40) polyadenylation signal (pA) was placed downstream of FRT site for selecting correct cassette exchange by RMCE (FIG. 1). Single site integration of one copy of the landing pad vector in the MCL was confirmed by southern blotting and targeted locus amplification (TLA) analyses (Cergentis). The MCL was grown in a protein-free medium consisting of HyQ PF (GE Healthcare Life Sciences) and CD CHO (Thermo Fisher Scientific) at a ratio of 1 :1 and supplemented with 1 g/L sodium carbonate (Sigma), 6 mM glutamine (Sigma), and 0.1 % Pluronic F-68 (Thermo Fisher Scientific) in a humidified Kuhner shaker (Adolf Kuhner AG) with 8% CO2 at 37°C. Routine subculture was conducted every 3 to 4 days by seeding cells at density of 3x10 ⁵ cells/mL in 15 mL of fresh medium in 125 mL shake flasks (Corning). Cell density and viability were determined by trypan blue exclusion method on Vi-Cell XR viability analysers (Beckman Coulter).

Construction of targeting vectors for expression of heparan sulfate modification enzymes

The targeting vectors containing single heparan sulfate modification gene or scaffold were synthesized by Genscript. A list of the genes used in this work and its corresponding accession number is included in Table 1. For all constructs, genes were derived from human, except for three of the HSPG (Glypican 1 , Perlecan 1 and Syndecan 1). These three HSPG genes were obtained from other animal sources as they have been previously successfully expressed in CHO cells by other studies in the art and had been shown to be decorated with heparan sulfate. In the present disclosure, the entire gene for each gene of interest (GOI) was expressed in CHO cells except for Perlecan, for which only domain 1 (containing the heparan sulfate attachment site) was expressed. To generate dual heparan sulfate modification gene constructs, the vector backbone with two GOI insertion sites was generated by Genscript. Restriction enzyme sites Mlul and Xhol were used to replace GOI1 while Mfel and EcoRI were used to replace GOI2 (FIG. 1A). For the triple heparan sulfate modification gene constructs, the inventors of the present disclosure gene synthesized a vector (Genscript) similar to the dual gene vector with the exception of the replacement of the pA with an IRES. Using this vector, the puromycin resistant gene was replaced with serglycin using EcorV and Noth The sequence of SpA was from the pcDNA3.1 (+) vector (Thermo Fisher Scientific) while the IRES sequence was obtained from a previous study in the art.

Table 1 : List of heparan sulfate modification enzymes and proteoglycan genes used in the study. Generating stable HS-producing cell lines via recombinase-mediated-cassette- exchange (RMCE)

The MCL were co-transfected with an appropriate targeting vector and a vector expressing FLPe using Amaxa SG Cell Line 4D-Nucleofector® X Kit Land program FF- 137 (Lonza). In each transfection, 5x10 ⁶ cells were transfected with 5 g of targeting plasmid vector and 5 pg of FLPe plasmid vector in circular format. The transfected cells were then re-suspended in 2 mL of PFM preloaded in 6-well suspension culture plates (NUNCTM) and incubated in static incubators (IncuSafe, Sanyo). At 24 h post- transfection, they were collected by centrifugation (100xg, 5 min) and re-suspended in 15 mL of protein-free medium in 125 mL shake flasks in the humidified Kuhner shaker (Adolf Kuhner AG) with 8% CO2 at 37°C. Four days later, the transfected cells were subjected to selection in the PFM containing Puromycin (Invivogen) at 20 pg/mL. Selection was continued for two weeks by passaging in the selection medium every 3 to 4 days. Stably transfected cell pools were deemed established when cell viabilities recovered over 95%.

Genomic integration analysis

To check for correct cassette recombination, genomic DNA (gDNA) was extracted from 5X10 ⁶ CHO cells using Purelink Genomic DNA Mini Kit (Thermo Fisher) according to manufacturer’s instructions. 100ng of the gDNA was used as a template for PCR using Platinum™ SuperFi II Green PCR Master Mix (Thermo Fisher). Primer sets flanking the 5’ and 3’ integration sites were used to check for successful integration of the transgene. Primers used in the junction PCRs are listed in Table 2. PCR products were visualized on 1% agarose gels stained with ethidium bromide. Table 2. List of primers for RT-PCR analysis

RNA extraction and reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was extracted from 5x10 ⁶ CHO cells using RNeasy mini kit (Qiagen) according to manufacturer’s instructions. The amount of RNA extracted was assessed by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific). For RT-PCR, 1 pg of total RNA from each sample was used as template for cDNA synthesis using Lunascript RT SuperMix Kit (New England Biolabs) according to manufacturer’s instructions. Primers specific to the different heparan sulfate modification enzymes were used to prime the cDNA for presence of the RNA. A list of primers used is given in Table 3. RT-

PCR products were separated on 1% agarose gel and visualized by ethidium bromide staining.

Table 3: List of primers for integration PCR check Fed-batch Culture for heparan sulfate production

To generate recombinant HS, cell lines expressing the different combinations of enzymes were seeded at 3x10 ⁵ cells/mL in 50 mL TPP® TubeSpin bioreactor tubes (Sigma Aldrich) containing 30 mL of protein-free media described above. EX-CELL Advanced CHO Feed 1 with glucose (Sigma Aldrich) at 10% of the culture volume was added to each TubeSpin on days 3, 5, 7, 9 and 11. To maintain sufficient glucose level, 3 g/L of glucose were supplemented when the glucose concentration in the culture medium drops below 3 g/L. Heparan sulfate in the culture medium was harvested on day 7 and day 14 by centrifugation at 3000 rpm for 15 mins and collecting the supernatant.

Single cell cloning of transfected CHO cells To isolate CHO cell clones for heparan sulfate composition analysis and yield, transfected CHO cells were diluted to a final concentration of 600 cells/ml in CloneMedia (Molecular Devices). 12ml of cell suspension were aliquoted into each petri dish and incubated with 8% CO2 at 37°C in static incubator (IncuSafe, Sanyo) for 14 days. Cell colonies formed were picked up and transferred into 24-well plates (NLINCTM) and allowed to grow till 50% confluent. Cells are then transferred into 50 mL TPP® TubeSpin bioreactor tubes (Sigma Aldrich) containing 5 mL of protein-free media described above.

Isolation of heparan sulphate from conditioned media

CHO conditioned media was vacuum filtered through a 0.2 pm membrane treated with 10 mill chondroitinase ABC (Merck), 10 mill neuraminidase (Merck), 1000 kunitz units DNAse I (Merck) and 500 units RNase IA (Merck) in the presence of 0.02% (w/v) sodium azide for 16 h at 37°C. Pronase (from streptomyces griseus, Merck) was added to a final concentration of 500 pg/mL and media incubated for a further 4 h at 37°C. Media was cooled to room temperature and heparan sulfate isolated using a HiTrap Capto Q column (5 mL; Cytiva) attached to an Akta Pure 25M FPLC system (Cytiva). 25 mL of media was applied to the column, then washed with 10 column volumes (C.V.) of 150 mM sodium chloride, 20 mM sodium phosphate monobasic, 20 mM sodium phosphate dibasic, pH 7.2, followed by 10 C.V. 200 mM sodium chloride. Heparan sulfate was eluted with 2M sodium chloride and subsequently desalted using a HiPrep 26/10 desalting column attached to an Akta Pure 25M FPLC system. Desalted heparan sulfate isolates were frozen and lyophilised to dryness prior to weighing.

Disaccharide compositional analysis

500 pg (1 mg/mL) of purified heparan sulfate samples were treated with 2 ml U of heparinase I, II and III (Iduron, U.K.) in digest buffer (100 mM sodium acetate, 1 mM calcium acetate, pH 7) for 24 h at 37°C. After 24 h, a second 2 mIU aliquot of hepainase I, II and III were added to each sample and the digest allowed to proceed for a further 24 h at 37°C. Once complete, samples were heated at 100°C for 5 min, cooled and disaccharides isolated via size exclusion chromatography using an Akta Pure 25M with Superdex Peptide 10/300 gl SEC column under isocratic flow (0.75 ml/min) in 200 mM ammonium bicarbonate. Isolated disaccharides were frozen and lyophilised twice to remove ammonium bicarbonate. Compositional analysis was performed similarly to a study known in the art such as Carnachan et a/.(Carbohydr Polym, 2016. 152: p 592- 597). Briefly, disaccharides were resuspended in 0.5 mL of water (pH 3.5), filtered (0.22 m) and 100 pL applied to a ProPac PA1 column (4 x 250 mm with 4 x 50 mm guard column. Thermo Fischer). Disaccharides were eluted using a salt gradient (buffer A = water, pH 3.5, buffer B = 2M sodium chloride, pH 3.5) at 1 mL/ min (40°C column temperature) using the following gradient: 0% B for 1 min, 0 to 35% B for 31 min, 35 to 65% B for 15 min, 100% B for 10 min, 0% B for 3 min). Eluted disaccharides were monitored at UV 232 nm and identified based on the elution points of known disaccharide standards (Iduron, U.K.). Percentage composition was calculated based on the division of the peak area of each disaccharide by the sum of all peak areas, multiplied by 100. Data represents mean ± S.D. of triplicate analysis of two independent digests.

Factor Xa assay

The factor Xa assay was carried out as described by a study known in the art (Zubkova et al., ACS chemical biology, 2018. 13(12): p. 3236-3242). Briefly, heparan sulfate samples were diluted in assay buffer (175 mM NaCI, 50 mM Tris, 7.5 mM EDTA, 0.1% [w/v] PEG 8000, pH 8.4) and aliquoted in triplicate (30 pL) into flat-bottomed, clear 96-well plates. The following pre-warmed (37°C) reagents were added at 2 min intervals: antithrombin III (0.015 IU; Provision Kinetics, USA), factor Xa (75 ng; Provision Kinetics, USA), factor Xa substrate (18.75 pg; Provision Kinetics, USA), acetic acid (20% (v/v)). After the addition of acetic acid, absorbance at 405 nm was measured using a Hidex Sense spectrophotometer. lU/mg values were calculated via standard curve (slope ratio) using the heparin USP standard.

Surface plasmon resonance

SPR solution competition was carried out as described by a study known in the art (Smith et al, Biomaterials, 2018. 184: p. 41-55). Briefly, heparin (Merck) was biotinylated at the reducing end by resuspended in 100 mM sodium acetate, pH 4.5 at 20 mg/mL with 50 mM alkoxyamine-PEG4-biotin (Thermo Fisher) and 10 mM aniline. After 48 h, the biotinylated heparin was desalted using a HiPrep 26/10 column, lyophilised and weighed. Series S streptavidin sensor chips (Cytiva) were coated with biotinylated heparin (2 pg/mL) in HSB-EP 0.05% buffer (150 mM sodium chloride, 10 mM HEPES, 3 mM EDTA, 0.05% [v/v] tween-20, pH 7.4) using the inbuilt immobilization program of a Biacore T200 instrument. One flow cell was immobilized with 40 response units (R.U.) of biotinylated heparin and another left blank to serve as a control surface. For solution competition experiments, heparan sulfate variants (10 pg/mL) were incubated with either FGF2 or FGFR1 lllb Fc (12.5 nM) in HBS-EP 0.1% buffer and applied to the heparin coated sensor chip using the following program: 120 s sample application, 700 s dissociation, 2 x 60 s regeneration. Chip regeneration was performed with high salt (2 M sodium chloride in HBS-EP 0.1 %). The same method was followed for HS-TG solution competition experiments, incubating HS-TG (5, 10 or 20 pg/mL) with BMP2 (12.5 nM), FGF2 (12.5 nM), FGFR1 II lb (12.5 nM), FGFR2 I lib (12.5 nM), PDGF- AB (12.5 nM), PDGF-BB (12.5 nM), SDF1a (100 nM), TGF 1 (100 nM) or VEGF s (12.5 nM). Data was recorded as a measure of response over time at 25°C (sensorgram). BIAevaluation software was used to determine affinity, affinity measurements were normalised to protein alone, then multiplied by 100 and subtracted from 100 to give percentage protein in solution. Data represents mean ± S.D. from three independent experiments. FIG. 9A to 9F show SPR sensorgrams from solution competition experiments and FIG. 10A to 101 show SPR sensorgrams from HS-TG solution competition experiments.

MSC proliferation

Human MSCs were maintained in maintenance media (DM EM (low glucose), 10% [v/v] foetal calf serum, 4 mM L-glutamine, 100 lll/mL penicillin and 100 pg/mL streptomycin) in 5% CO2 at 37°C and used at passage 5 for proliferation assays. Cells were seeded at 5000 cells/cm ² in 24-well tissue culture plastic plates and allowed to attach for 24 h prior to treatment with FGF-2 (1.25 ng/mL) or heparan sulfate variants (50 pg/mL) in maintenance media. For treatments, FGF2 ± HS were incubated for 10 min at room temperature in 20 pL PBS prior to adding to the cell media. For inhibition experiments, PD173074 in DMSO was combined with PBS, heparan sulfate, and FGF2 at concentrations of 25, 50 or 100 nM. FGFR1 blocking antibody IMBR1 was added at the stated dilutions (1 :1000 or 1 :2000) to treatments prior use. Cells were then cultured for 8 days (with a second treatment at day 3) and harvested at day 8 for viability analysis using a Guava EasyCyte instrument. Total number of viable cells were normalised to no treatment and presented as relative viable cell number. Data represents mean ± S.D. of three independent experiments.

FGFR1 : FGF2 and FGFR2: FGF2 coimmunoprecipitation experiments

Co-immunoprecipitation of FGFR1 : FGF2: heparan sulfate complexes was performed as follows. Briefly, FGFRIa lllb Fc chimera (125 nM) or FGFR2a lllb Fc chimera was bound to protein A/G agarose beads at 4°C for 1 h. FGF2 (250 nM) ± HS- TG (12.5, 25 or 50 pg/mL) were added to the beads and incubated at room temperature for 1 h. Beads were subsequently washed with 1 mL PBS x 5, then boiled in Laemmli buffer prior to loading into NuPAGE 8-12% SDS PAGE gels. Gels were run at 150 V for 70 min, and protein was transferred to nitrocellulose membranes using wet transfer apparatus at 100 V for 90 min. FGFR1 and FGF2 were detected using Mouse antiHuman FGFR1 (1 pg/mL in 5% BSA) and Goat anti-Human FGF2 (1 pg/mL in 5% BSA) respectively. HRP-conjugates secondary antibodies were used for detection, and membranes imaged using an Invitrogen iBright imaging system (Thermo-Fisher Scientific) after treatment with SuperSignal West Pico chemiluminescent substrate (Thermo-Fisher Scientific). Blots are representative of three independent experiments, and densitometry constitutes mean ± S.D. fold change in FGF-2 versus FGF2: FGFR1 or FGF2: FGFR2 alone of three independent experiments.

RESULTS

Design of recombinase-mediated-cassette-exchange (RMCE) to screen for heparan sulfate modifying enzymes with impact on heparan sulfate bioactivity and yield

To study the impact of heparan sulfate modification enzymes in generating heparan sulfate of diverse structures, different heparan sulfate modification enzymes were expressed either individually or in combination in the CHO K1 master cell line (MCL) of the present disclosure via RMCE based targeted integration (FIG. 1). The MCL contains a single landing pad vector, which was confirmed by a previous study in the art by Southern blotting and targeted locus amplification (TLA) analysis (data not shown). The landing pad vector contained an eGFP gene and a Zeocin selection marker flanked by a wild type flippase recognition target (FRT) and its mutant counterpart (FRT3). An impaired puromycin resistant gene lacking a start codon ((ATG-) Puro) was placed downstream of the FRT site. The chimeric promoter (ChiP) upstream of the FRT3 site drove the transcription of the GFP and Zeo resistant gene. CAP independent translation of the Zeo resistant gene was driven by an encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) and terminated by a SV40 polyadenylation signal (pA) upstream of the FRT site. The design of the landing pad vector ensures that the impaired puromycin is only expressed after successful activation by RMCE (only applicable for triple gene integration experiments).

Targeting vectors were designed to be promoterless and contained either one or two heparan sulfate modifying enzymes and, in some combinations, included the scaffold protein serglycin (FIG. 1). For the single and dual enzyme vectors, the first gene in the vector is the puromycin resistant gene and thus successful integration relies on the promoter trap to activate the puromycin in the targeting vector to survive selection. These vectors have a pA just before the FRT site to stop the activation of the (ATG-) puro in the landing pad. For the triple gene vectors, the puromycin resistant gene was replaced with a serglycin gene. For these constructs, an additional wild-type ECMV IRES was placed downstream of GOI 2 for activating the (ATG-) puromycin after successful replacement of the GFP-Zeo cassette in the MCL via RMCE.

RMCE was conducted by co-transfection of a specific targeting vector together with a vector expressing the enhanced flippase (FLPe) and selected for with puromycin to obtain stably transfected pools. As the targeting vectors do not contain a promoter, random integration of the vector into the genome will not activate the carried genes. Successful integration of the cassette into the landing pad will result in the loss of GFP expression, which serves as a rapid mode of identifying successful integrant. After selection, the recovered cell lines were validated for successful targeted integration by extracting total RNA and genomic DNA to check for the presence of gene specific RNA via RT-PCR (FIG. 7) as well as for the presence of correct integration into the CHO genome via junction PCR (FIG. 8). The genomic analysis of the present disclosure indicated that all cell lines produced using the RMCE method have correct gene integration and the transcripts present in the cells.

Analysis of heparan sulfate from nine CHO lines expressing individual heparan sulfate biosynthetic enzymes

FIG. 2A to 2F shows the characterisation of heparan sulfate variants isolated from the media of CHO lines transfected with one or two heparan sulfate biosynthetic genes of interest. Each modified cell line was grown for either 7 days or 14 days under fed- batch conditions for heparan sulfate isolation. These time points were chosen as cells reach the maximal viable cell density at day 7, and culture viability starts to drop below 50% at day 14. Heparan sulfate was successfully isolated from CHO cell conditioned media (CM; day 7 and day 14) using anion exchange chromatography, then subsequently desalted, and weighed. Heparan sulfate was isolated from 25 mL of CM, with total mass of heparan sulfate isolated varying between cell lines (FIG. 2A). Interestingly, expression of any of the nine chosen HS biosynthetic enzymes increased the overall yield of heparan sulfate at day 7 and day 14 over the parental CHO K1 line, with NDST2 producing the highest mass of heparan sulfate at day 7 (-97.2 pg/mL media; FIG. 2A) and NDST1 producing the highest mass at day 14 (-105.9 pg/mL media; FIG. 2A). Increasing culture time from day 7 to day 14 did not result in a two-fold increase in heparan sulfate mass, suggesting other factors such as decreasing cell viability may lead to a decrease in the rate of accumulation of heparan sulfate in CM beyond day 7.

Next, the inventors of the present disclosure assessed the composition of each heparan sulfate variant using enzymatic depolymerisation followed by strong anion exchange HPLC (FIG. 2B). Expression of any of the nine chosen heparan sulfate biosynthetic enzymes resulted in a marked change in the composition of heparan sulfate at day 7 and day 14 (FIG. 2Bi-ii). As expected, NDSTs 1 and 2 both resulted in a notable increase in the overall percentage of AUA-GIcNS residues, (NDST1 - day 7 = 63.64% and day 14 = 59.62%; NDST2 - day 7 = 75.97% and day 14 = 80.84%). 6OST1 , 2 and 3 also resulted in increases in the total percentage of 6-O-sulphated disaccharides, predominantly AUA-GlcNAc6S (6OST1 = 41.93%, 6OST2 = 20.44% and 6OST3 = 42.91% at day 7) but also AUA-GlcNS6S (6OST1 = 30.54%, 6OST2 = 17.51% and 6OST3 = 21 .49% at day 7). Expression of 3OST 1 or 3OST5 did not markedly affect the composition of heparan sulfate. Unfortunately, 3-O-sulphated disaccharide standards are not currently commercially available, so it was not possible to quantify their presence in the present disclosure using the chosen method of analysis of the present disclosure. Expression of either GLCE or 2OST had no notable effect of the overall composition of heparan sulfate versus the parental cell line at day 7 and day 14 (FIG. 2B-C).

The inventors of the present disclosure next sought to assess the binding affinity of each heparan sulfate variant toward FGF2, a potent mitogen and essential survival factor for MSCs. Here, the inventors of the present disclosure used SPR solution competition, pre-complexing 10 pg/mL of each heparan sulfate variant with 12.5 nM of FGF2 before applying to a heparin coated SPR chip (FIG. 2C). All heparan sulfate variants were able to bind and subsequently prevent FGF2 from binding to the heparin- coated surface (FIG. 2D); heparan sulfate derived K1 (day 7 - 84.2%, day 14 - 85.9%), 3OST1 (day 7 - 84.2%, day 14 - 77.8%) and 3OST5 (day 7 - 85.4%, day 14 - 77.6%) lines displayed the highest affinity toward FGF2, followed by NDST 1 (day 7 - 61 .7%, day 14 - 54.2%) and NDST2 (day 7 - 60.9%, day 14 - 68.2%), then 6OST2 (day 7 - 59.4%, day 14 - 86.8%). GLCE (day 7 - 53.6%, day 14 - 40.5%), 2OST (day 7 - 31 .6%, day 14 - 25.7%), 6OST1 (day 7 - 14.5%, day 14 - 8.1 %) and 6OST3 (day 7 - 26.2%, day 14 - 22.3%). Heparan sulfate variants all displayed lower affinity toward FGF2 (FIG. 2C). These data suggest that subtle changes in heparan sulfate domain structure and composition may play an important role in FGF2 binding affinity.

Finally, the inventors of the present disclosure assessed the biological activity of the heparan sulfate variants using an MSC proliferation assay. As expected, MSCs cultured with exogenous FGF2 (1.25 ng/mL) displayed an increase in the relative number of viable cells, relative to the untreated control (2.04-fold, FIG. 2D). Interestingly, all heparan sulfate variants enhanced MSC proliferation over 2-fold after 8 days of culture, except for day 14 CHO K1 heparan sulfate (which saw a reduction in viable cells, FIG. 2D).

Analysis of heparan sulfate from four CHO lines expressing two heparan sulfate biosynthetic enzymes

The inventors of the present disclosure subsequently generated CHO lines expressing two heparan sulfate biosynthetic enzymes, based on the screen of the present disclosure of heparan sulfate derived from CHO lines expressing individual enzymes. Each construct contained NDST2 and one other gene. NDST2 was chosen because NS domains serve as substrates to other sulphotransferases, and to ascertain if two genes will synergistically result in further alterations in the composition of the isolated HS.

The mass of heparan sulfate isolated per mL of CM was highest for GLCE- NDST2 cell line, with ~63 pg/mL and -115 pg isolated at days 7 and 14 respectively (FIG. 2E). 6OST1-NDST2, 6OST2-NDST2 and 6OST3-NDST2 CM yielded similar quantities of heparan sulfate at days 7 and 14 (45, 52 and 39 pg/mL at day 7 and 95, 99 and 90 pg/mL at day 14, respectively: FIG. 2E). Compositional analysis revealed GLCE- NDST2 heparan sulfate was composed predominantly of AUA-GIcNS residues at days 7 (70.28%) and 14 (73.82%), whereas the three 6OST variants displayed variable increases in AUA-GlcNS6S (day 7 - 6OST1 - 12.5%, 6OST2 - 9.8%, 6OST3 - 9%; day 14 - 6OST1 - 22.6%, 6OST2 - %, 6OST3 - 16%) and AUA2S-GlcNS6S (day 7 - 6OST1

- 6.2%, 6OST2 - 27.3%, 6OST3 - 8.4%; day 14 - 6OST1 - 12.9%, 6OST2 - %, 6OST3

- 19.6%) residues (FIG. 2F). Affinity toward FGF2 was highest for 6OST2-NDST2 HS at days 7 and 14 (97.3% and 79.9%), followed by GLCE-NDST2 (76.1% and 75.4%), 6OST3-NDST2 (69.9% and 72.4%) and finally 6OST1-NDST2 heparan sulfate (59.1% and 52.8%), although all variants were able to sequester >50% of FGF2 into solution (FIG. 2G). The inventors of the present disclosure then sought to establish if the heparan sulfate variants could bind the cognate receptor of FGF2, FGFR1 by using an Fc chimera of the 11 lb splice variant. All heparan sulfate variants from cell lines expressing two genes bound FGFR1 111 b with high affinity (FIG. 2H; all > 88%), which contrasted with CHO K1 heparan sulfate, were only day 7 heparan sulfate isolates were able to effectively bind FGFR1 lllb (FIG. 2H; day 7 - 87.3%, day 14 - 11.7%). Finally, all two gene heparan sulfate variants were able to enhance MSC proliferation to varying degrees. Day 7 GLCE-NDST2, 6OST1-NDST2, 6OST2-NDST2 and 6OST3-NDST2 heparan sulfate variants were able to enhance MSC proliferation by 1.48-fold, 1.26-fold, 1.33-fold, and 1.21 -fold respectively, and day 14 variants by 1.47-fold, 1.51 -fold, 2.35-fold, and 1.45- fold respectively (FIG. 2I). Taken together, these data indicate that the expression of two heparan sulfate biosynthetic genes can lead to synergistic changes in heparan sulfate composition, which subsequently enhance FGF2 and FGFR1 binding affinity and result in greater biological activity.

Analysis of heparan sulfate from four CHO lines expressing heparan sulfate proteoglycans

FIG. 3A to 3D shows the characterisation of heparan sulfate variants isolated from the media of CHO lines transfected with HSPGs. The inventors of the present disclosure next sought to increase the total mass of heparan sulfate produced by generating four cell lines overexpressing one of the following HSPGs: syndecan 1 , glypican 1 , domain 1 of perlecan or serglycin. The mass of heparan sulfate isolated per mL of CM markedly increased in syndecan (day 7 - 131 pg/mL, day 14 - 136 pg/mL), glypican (day 7 - 91.6 pg/mL, day 14 - 165 pg/mL), perlecan (day 7 - 165 pg/mL, day 14 - 190 pg/mL) and serglycin (day 7 - 130 pg/mL, day 14 - 258 pg/mL) expressing cell lines (FIG. 3A). The composition of each heparan sulfate variant was subsequently assessed, with all possessing a largely similar composition (FIG. 3B). Notably, heparan sulfate isolated from syndecan-1 (day 7 and 14) and serglycin (day 7) lines was more highly sulphated, with increases in AUA2S-GlcNS (syndecan day 7 = 18.13%, day 14 = 24.82%; serglycin day 7 = 24.73%) and AUA2S-GlcNS6S (syndecan day 7 = 23.94%, day 14 = 25.28%; serglycin day 7 = 21.37%) residues (FIG. 3B). Analysis of FGF2 binding via SPR solution competition indicated all heparan sulfate variants possessed similar affinity toward FGF2 (day 7 - 82-88%; day 14 - 77-85%), comparable to heparan sulfate derived from the CHO K1 line (FIG. 3C). This suggests increasing heparan sulfate biosynthesis has not compromised FGF2 binding affinity. Finally, the bioactivity of each heparan sulfate variant was assessed using the MSC proliferation assay. All heparan sulfate variants performed similarly, resulting in 1.57-fold to 1.79-fold increases in viable cells over control (FIG. 3D). Taken together, these data suggest the overall mass of heparan sulfate produced by CHO lines can be increased by overexpressing HSPG core proteins without negatively impacting structure, FGF2 binding affinity or bioactivity.

Analysis of heparan sulfate from four CHO lines expressing two heparan sulfate biosynthetic enzymes and serglycin

FIG. 4A to 4E shows the characterisation of heparan sulfate variants isolated from the media of CHO lines transfected with serglycin and two heparan biosynthetic enzymes. For the final cell lines, the inventors of the present disclosure combined the previously selected combinations of two heparan sulfate biosynthetic enzymes with serglycin to generate four cell lines expressing three genes. The inventors of the present disclosure hypothesised that these cell lines would produce heparan sulfate with similar composition, binding affinity, and bioactivity as the two gene equivalents, and that the addition of serglycin would increase the total mass of heparan sulfate produced.

Interestingly, the mass per mL of heparan sulfate isolated from day 7 CM did not increase substantially (36 to 63 pg/mL), however, mass per mL of heparan sulfate isolated at day 14 markedly increased for serglycin-GLCE-NDST2, serglycin-6OST1- NDST2 and serglycin-6OST3-NDST2 lines (126 pg/mL, 183 pg/mL, and 253 pg/mL respectively: FIG. 4A). Unexpectedly, the mass/ mL of heparan sulfate isolated from serglycin-6OST2-NDST2 CM was lower than the CHO K1 line (36 pg/mL and 78 pg/mL: FIG. 4A). Interestingly, the addition of serglycin resulted in marked changes in the composition of heparan sulfate isolated from CHO lines expressing 6OST1 , 2 or 3. The resultant heparan sulfate chains were primarily composed of AUA-GIcNS (day 7 - 6OST1 - 8.4%, 6OST2 - 26.6%, 6OST3 - 6.8%; day 14 - 6OST1 - 17.2%, 6OST2 - 39.8%, 6OST3 - 15%) and AUA-GlcNS6S (day 7 - 6OST1 - 84%, 6OST2 - 54.6%, 6OST3 - 84.2%; day 14 - 6OST1 - 73.5%, 6OST2 - 29%, 6OST3 - 75.9%) residues (FIG. 4B). This observation is significant, suggesting the core protein of HSPGs can influence the composition of the heparan sulfate chains they harbour. Serglycin-GLCE- NDST2 heparan sulfate chains were compositionally similar to the dual gene equivalents, predominantly composed of AUA-GIcNS residues (day 7 - 73.4%, day 14 - 74.3%: fig. 4B). A small reduction in overall sulphation was observed from day 7 to day 14 HS isolates, consistent with observations in our single and dual gene lines (FIG. 4B). This is consistent with observations in a previous study known in the art, which suggested sulfation levels in CHO heparan sulfate decrease as culture time increased. FGF2 affinity as determined by SPR solution competition assays was high for all heparan sulfate variants, with each able to sequester 80% or greater FGF2 into solution (FIG. 4C). Interestingly, all three gene heparan sulfate variants displayed lower affinity toward FGFR1 lllb (day 7 - 45 to 55%; day 14 - 18 to 42%) than two gene heparan sulfate variants (FIG. 4D). Of the four heparan sulfate variants, those derived from cell lines expressing 6OSTs displayed higher affinity (specifically, those isolated from day 7 culture supernatant, 50 to 55%; FIG. 4D), which agrees with a previous study in the art in terms of the requirement of 6-O-sulphation for effective binding of heparan sulfate to FGFR1. Finally, the inventors of the present disclosure used the MSC proliferation assay to assess the bioactivity of each heparan sulfate variant (FIG. 4E). All heparan sulfate variants enhanced the proliferation of MSCs, with day 7 serglycin-6OST2-NDST2, day 14 serglycin-6OST1-NDST2, and day 14 serglycin-6OST3-NDST2 heparan sulfate able to surpass the effect of 1.25 ng/mL of FGF2 (1.64-fold, 1.58-fold, and 1.58-fold vs. 1.43- fold increase, respectively; FIG. 4E). All variants resulted in at least a 20% increase in the total number of viable cells (FIG. 4E).

Taken together, these data suggest CHO-derived recombinant heparan sulfate variants could be used as MSC media bio-additives. It also highlights the simultaneous expression of serglycin and sulphotransferases may synergistically influence the composition of heparan sulfate.

Scaled production and isolation of HS-TG

FIG. 5A to 5F shows the characterization of HS-TG. After screening four triple gene heparan sulfate variants, serglycin-6OST3-NDST2 was selected (based on mass of heparan sulfate produced) for scaled production and named ‘HS-TG’. A total of 250 mg of heparan sulfate was isolated from ~1.2 L of CM after 14 days of culture, representing a significant increase in heparan sulfate yield over the CHO K1 line (217 pg/mL > 80 pg/mL, FIG. 5A). Disaccharide analysis revealed a similar composition to the original small batch of serglycin-6OST3-NDST2, predominantly AUA-GlcNS6S residues (69%; FIG. 5B). This again contrasts heparan sulfate isolated from the dual gene equivalent 6OST3-NDST2, which is composed of a much lower percentage of AUA- GlcNS6S residues (16%; FIG. 6B). Though highly sulphated, HS-TG should possess negligible anticoagulant activity due to the lack of expression of 3OST 1 and 3OST5 in CHO cells. This was confirmed using a chromogenic factor Xa activity assay, with HS- TG registering 1 < lll/mg anti-factor Xa activity (FIG. 5Ci-ii), much lower than heparin (>140 lU/mg). Next, the binding affinity of HS-TG toward various heparan sulfate-binding proteins was assessed via SPR solution competition (FIG. 5D). HS-TG (5, 10 or 20 pg/mL) was incubated with BMP2 (12.5 nM), FGF2 (12.5 nM), FGFR1 lllb (12.5 nM), FGFR2 lllb (12.5 nM), PDGF-AB (12.5 nM), PDGF-BB (12.5 nM), SDF1a (100 nM), TGFpi (100 nM) or VEGFies (12.5 nM) and applied to a heparin-coated SPR chip. HS- TG displayed high affinity for FGF2, PDGF-AB, PDGF-BB and TGFpi , middling affinity for BMP-2, SDF1a and VEGFies, and lower affinity for FGFR1 lllb and FGFR2 lllb (FIG. 5D). It is interesting to note that HS-TG displayed higher affinity toward FGFR1 lllb than FGFR2 lllb (FIG. 5D). The inventors of the present disclosure next assessed the ability of HS-TG to enhance the thermal stability of FGF2 using differential scanning fluorimetry (FIG. 5Ei-ii). Addition of HS-TG at increasing concentrations (200, 400 or 600 pg/mL) resulted in a dose dependent increase in the thermal stability of FGF2 (10 pM), as indicated by a shift in peak fluorescence of the melt curve to a higher temperature (FIG. 5Ei). The melt temperature of FGF2 (56.66°C) was increased to 58.75°C, 61 .41 °C and 64°C with HS-TG at 200, 400 or 600 pg/mL respectively (FIG. 5Eii).

SPR gave clear indication that HS-TG can bind FGF2, FGFR1 , and FGFR2, so the inventors of the present disclosure next sought to ascertain if HS-TG is able to enhance the formation of FGF2: FGFR1 , and FGF2: FGFR2 receptor complexes using co-immunoprecipitation (FIG. 5F). FGFRIa lllb or FGFR2a lllb Fc chimeras were coated on protein A/G beads and incubated with FGF2 ± at increasing concentrations of HS-TG (12.5, 25 or 50 pg/mL). Alone, both FGFR1 and FGFR2 bound minimal FGF2, an expected result as heparan sulfate was reported in previous study in the art to play an integral role in the formation of FGF2: FGFR1 and FGF2: FGFR2 complexes. HS-TG markedly increased the amount of FGF2 bound to FGFR1 (7.65-fold, 7.8-fold, and 6.76- fold at 12.5, 25 and 50 pg/mL respectively: FIG. 5Fi-ii). Addition of HS-TG also increased the amount of FGF2 bound to FGFR2, although to a lesser extent than FGFR1 (2.56- fold, 2.33-fold, and 2.43-fold at 12.5, 25 and 50 pg/mL respectively: FIG. 5Fiii-iv). These data indicate HS-TG could enhance formation of FGF2:FGFR1 complexes over FGF2:FGFR2 complexes (FIG. 5F).

Figure 6A to 6E shows characterization of the bioactivity of HS-TG and isolation of HS-TG from multiple clones. The inventors of the present disclosure next assessed the bioactivity of HS-TG using the MSC proliferation assay. Increasing doses of HS-TG resulted in a dose-dependent increase in the total number of viable cells after 8 days of culture (FIG. 6A). At a dose of 25 pg/mL, HS-TG matched the proliferative effect of FGF2 (1.25 ng/mL) and surpass it at doses of 50 and 100 pg/mL. HS-TG resulted in 1.35-fold (6.25 pg/mL), 1.6-fold (12.5 pg/mL), 1.85-fold (25 pg/mL), 2-fold (50 pg/mL) and 2.18- fold (100 pg/mL) increases in viable cells over no treatment (FIG. 6A), highlighting the potential of HS-TG as a potent media additive for MSC expansion. To confirm the proliferative effect of HS-TG is mediated through the FGF2: FGFR1 signalling pathway, the inventors of the present disclosure performed two FGFR1 inhibition experiments. In the first, the inventors of the present disclosure employed an FGFR1 blocking antibody, IMBR1 , previously developed in-house as an anti-cancer therapeutic (FIG. 6Bi). IMBR1 binds the heparin-binding domain of FGFR1 , preventing heparan sulfate from binding FGFR1 preventing formation of the ternary FGFR1 : FGF2: heparan sulfate signalling complex. IMBR1 was added at 1 :1000 and 1 :2000 dilutions and resulted in a dosedependent reduction in the proliferative effects of FGF2, HS-TG and a combination of FGF2 + HS-TG (FIG. 6Bi). Notably, the addition of IMBR1 (1 :1000) to cells cultured with HS-TG resulted in a reduction in viable cells from 2.14-fold to 0.86-fold relative to the no treatment control (FIG. 6Bi). IMBR1 (1 :1000) also inhibited FGF2 (1.68-fold to 1.28-fold) and FGF2 + HS-TG (2.36-fold to 1.31 -fold), although to a lesser extent. It is possible that higher concentrations of IMBR1 would result in greater inhibition of MSC proliferation in FGF2 and FGF2 + HS-TG conditions. In the second inhibition experiment, the inventors of the present disclosure used an FGFR1 RTK small molecule antagonist (PD173074), which was able to inhibit the proliferation-enhancing effects of FGF2, HS-TG and a combination of FGF2 and HS-TG (FIG. 6Bii). This inhibition increased at doses of 25, 50 and 100 nM, with 100 nM reducing MSC proliferation below the basal levels observed in the no treatment control for FGF2 (from 1.75-fold at 0 nM to 1.35-fold, 0.97-fold, and 0.73-fold at 25, 50 and 100 nM respectively) and HS-TG (from 1.92-fold to 1.48-, 1.29-, and 0.86-fold at 0, 25, 50 and 100 nM respectively), and markedly decrease the proliferative effect of FGF2 and HS-TG combined at 100 nM (from 2.03-fold to 1.22-fold at 100 nM; fig. 6Bii). Taken together, these data suggest HS-TG enhances MSC proliferation through the FGFR1 : FGF2 signalling cascade and could be used to replace exogenous FGF2 in novel MSC media formulations.

Single cell cloning of CHO HS-TG cells

The inventors of the present disclosure next sought to determine if a targeted integration approach can produce CHO cell clones with comparable heparan sulfate yields, composition, and function, by isolating 10 clones from the cell line from which HS- TG was isolated (serglycin-6OST3-NDST2; FIG. 6C-E). Heparan sulfate isolated from the CM of these 10 clones ranged from 150 pg/mL (clone 44) to 195 pg/mL (clone 15), although the remaining 8 clones ranged between 162 and 183 pg/mL (FIG. 6C). Disaccharide analysis revealed all heparan sulfate variants to be compositionally similar, with all predominantly composed of AUA-GlcNS6S, (between 71 % [clone 44] and 78% [clones 32 and 38]), with AUA-GIcNS contributing to the remaining variability in composition (from 9% [clone 1] to 21% [clone 44]; FIG. 6D). The inventors of the present disclosure then used the MSC proliferation assay to determine the bioactivity of HS derived from each clone. Again, the effects were reasonably consistent across all clones, ranging from 2.28-fold (clone 1) to 2.86-fold (clone 23) increases in viable cell numbers relative to no treatment. The consistency between clones (all produce > 150 pg HS/ mL conditioned media, all possess >70% AUA-GlcNS6S residues, and all result in > 2.2-fold increase in viable MSCs over no treatment control) give clear indication that a targeted integration approach results in cell lines expressing similar levels of heparan sulfate with consistent and comparable composition and activity. This is crucial for mass production of heparan sulfate with minimal batch to batch variability and consistent functionality.

Translation of heparan sulfate into commercial or clinical settings is hindered by the animal-derived nature of commercially available sources. A second factor is the highly heterogeneous composition and structure of heparan sulfate, which results in an ill- defined mixture of polysaccharides of various molecular weights, structures, and compositions. Here, the inventors of the present disclosure used a metabolic engineering approach to systematically generate several transgenic CHO cell lines capable of synthesizing heparan sulfate of different composition. This was achieved using targeted integration technology, which permits overexpression of genes involved in heparan sulfate biosynthesis to generate structurally diverse heparan sulfate.

Targeted integration enables the inventors of the present disclosure to overcome the positional effects of random integration and thus minimize clonal variations, due to single copy insertion of heparan sulfate biosynthetic genes. This results in consistent expression levels of heparan sulfate biosynthetic enzymes within the Golgi, in turn resulting in the generation of similar heparan sulfate structures across multiple clones and eschewing the need for clonal selection. Together with the use of multi-cistronic vectors, the inventors of the present disclosure were able to engineer one, two or three genes from the heparan sulfate biosynthesis pathway simultaneously to generate a diverse range of heparan sulfate structures. Moreover, the inventors of the present disclosure have shown that expressing several heparan sulfate-relevant genes in a single construct can synergistically influence the composition, binding properties, and bioactivity of heparan sulfate, providing greater control over structure and function.

Expressing individual heparan sulfate biosynthetic enzymes resulted in modest changes in heparan sulfate structure and activity, specifically with heparan sulfate derived from CHO lines expressing NDST1 and 2, and 6OST1 , 2 and 3. Compositional changes due to expression of 3OST 1 and 3OST5 were not readily determinable by our chosen analysis method due to the lack of commercial standards for 3-O-sulphated disaccharides. Expression of GLCE and 2OST failed to yield any significant increases in 2-O-sulphation, it is possible that both would need to be expressed simultaneously to enhance 2-O-sulphation, as 2OST preferentially acts on iduronic acid residues ⁴³ . Additionally, enzymatic depolymerisation yields unsaturated uronic acids, precluding their identification. This prevented the inventors of the present disclosure from assessing any increases in iduronic acid content in heparan sulfate derived from GLCE-expressing CHO lines.

The expression of two heparan sulfate biosynthetic genes in one construct resulted in further changes in heparan sulfate composition, binding affinity, and bioactivity. The choice of the inventors of the present disclosure to consistently include NDST2 was made due to reasons previously explained: NDSTs generate the NS domains within heparan sulfate, which serve as substrates for C5-epimerase and the O- sulphotransferases, such as 2OST, the 6OSTs and the 3OSTs. The inventors of the present disclosure also selected NDST2 over NDST 1 as it resulted in a greater increase in N-sulphation. By enriching CHO heparan sulfate with NS domains through expression of NDST2, the inventors of the present disclosure hypothesised that 6-O-sulphation could be synergistically increased by also expressing one of the three 6OSTs. The result was an increased abundance of the disulphated disaccharide AUA-GlcNS6S in heparan sulfate isolated from these cell lines. The inventors of the present disclosure also hypothesized that constructs containing NDST2 and GLCE would result in an increase in iduronic acid residues, which could subsequently serve as substrates for endogenous 2OST. This was not the case, as the inventors of the present disclosure did not observe substantial increases in the presence of 2-O-sulphated disaccharides. Additionally, the method of the present disclosure of compositional analysis through enzymatic depolymerisation precludes the identification of uronic acid residues, as mentioned by previous study in the art. It is likely that simultaneous expression of NDST2, GLCE and 2OST would be required for a substantial increase in 2-O-sulphation. Heparan sulfate isolated from the four cell lines displayed high affinity toward FGF2 and its cognate receptor, FGFR1 , and subsequently enhanced bioactivity. This gives clear indication that altering heparan sulfate composition by expressing various components of the heparan sulfate biosynthetic pathway can result in favourable changes in heparan sulfate activity. This has also been highlighted in past studies in the art, focusing on the expression of NDST2 and 3OST1 , or 3OST1 and 6OST1 to generate recombinant heparin variants.

Ensuring production costs are met by sufficient product yield is an important factor in the production of biologies such as antibodies or recombinant proteins. The biosynthesis of heparan sulfate is far more complex than that of proteins and overexpressing any one of the many heparan sulfate biosynthetic genes is unlikely to result in markedly increased yield. 3’-phosphoadenosine-5’-phosphosulphate (PAPS) and uridine diphosphate (UDP) sugars are the key substrates in heparan sulfate biosynthesis, both are utilised in multiple biosynthetic pathways and are rate-limiting factors which influence heparan sulfate yield. The inventors of the present disclosure chose to overexpress various HSPGs to establish if this would result in increased heparan sulfate/ GAG biosynthesis, and to identify if an increase in the total mass of heparan sulfate produced would result in alterations in the overall composition due to rate-limiting factors such as insufficient PAPS. Previous studies in the art have investigated the expression of serglycin in HEK293 cells to produce recombinant heparin. Here, the inventors of the present disclosure found expression of serglycin enhanced the total mass of heparan sulfate produced by CHO cells by ~2.5-fold, without resulting in any substantial changes in sulphation, binding affinity, or bioactivity. Expression of domain 1 of perlecan, also resulted in increases in the total mass of heparan sulfate isolated from cell culture supernatants, an expected result as perlecan is a secreted HSPG.

The inventors of the present disclosure then chose to express three genes in one construct, composed of two biosynthetic enzymes and serglycin, which to the knowledge of the inventors of the present disclosure has not been previously attempted. Serglycin was chosen to enhance heparan sulfate yield, and the previously selected combinations of two heparan sulfate biosynthetic enzymes were also included in the construct. Strikingly, the presence of serglycin influenced the composition of heparan sulfate, resulting in a substantial enrichment in disulphated residues in triple gene constructs containing 6OST isoforms over the equivalent dual gene constructs. Serglycin is typically expressed highly in mast cells, where it serves as an intracellular HSPG harbouring heparin chain in secretory granules. The inventors of the present disclosure do not yet know if this phenomenon is unique to serglycin, perhaps in relation to serglycin’s function as an intracellular HSPG harbouring highly sulphated GAG chains, or if substituting serglycin with other HSPGs would result in similarly dramatic shifts in heparan sulfate chain sulphation. The enrichment in heparan sulfate sulphation in heparan sulfate derived from these cell lines translated into enhanced binding affinity toward FGF2, and subsequent increases in biological activity (as measured by MSC proliferation).

The investigation of the present disclosure into producing the selected recombinant heparan sulfate variant, HS-TG, at increased scale was successful, producing similar yields of heparan sulfate with similar structure and composition. HS- TG can interact with other heparan sulfate-binding proteins, including therapeutically relevant pro-regenerative factors such as VEGF165 and BMP2. This indicates HS-TG could have applications beyond media formulation and could be used to replace porcine- derived heparan sulfate in applications such as bone repair, angiogenesis, and cartilage repair. The inventors of the present disclosure also confirmed that the likely mechanism underpinning the effect of HS-TG on MSC proliferation is through enhancing formation of FGFR1 : FGF2 complexes, consequently activating the FGFR1 : FGF2 signalling cascade. Interestingly, whilst HS-TG enhanced formation of FGFR2: FGF2 complexes, the effect was not as pronounced as that observed with FGFR1 : FGF2. Preferencing the proliferation-promoting FGFR1 : FGF2 pathway over the differentiation-promoting FGFR2: FGF2 pathway further highlights the suitability of HS-TG as an MSC media additive for cell expansion. Further investigation of this mechanism is the target of more in-depth studies into the effects of HS-TG on MSC biology, expansion, and long-term culture in vitro. The function of heparan sulfate as a co-receptor is well known, and not limited to FGFR1 : FGF2, other well studied examples include VEGFR2: VEGF and others. These signalling cascades could also be enhanced with CHO-derived recombinant heparan sulfate variants for therapeutic applications.

Finally, the inventors of the present disclosure wanted to test the capability of the targeted integration platform of the present disclosure to generate CHO cell clones that produce heparan sulfate of comparable yield, composition, and function. Mass and disaccharide analysis of heparan sulfate isolated from 10 different clones showed that the heparan sulfate isolated was consistent in yield, composition, and bioactivity. This gives clear indication that the targeted integration approach can reduce clonal heterogeneity in heparan sulfate biosynthesis and enables the production of recombinant heparan sulfate variants with consistent batch yield, composition, and function without the need for isolating clones. This consistency reduces the time and effort required to screen hundreds of clones, thereby saving time and resources in the cell line development process.

The ability to tune the structure, and subsequently function of heparan sulfate derived from transgenic cell lines offers a great opportunity to tailor bespoke heparan sulfate variants for specific applications. Here, the inventors of the present disclosure chose to investigate the potential of engineering and using recombinant CHO-derived heparan sulfate as an MSC media adjuvant as proof of principle, though the technology could be used to generate heparan sulfate variants for a much wider range of applications. These could include bone repair, angiogenesis, cartilage regeneration, pluripotent stem cell culture and others.

In conclusion, the inventors of the present disclosure have demonstrated that the composition and subsequent function of heparan sulfate can be altered by using metabolic engineering to introduce copies of heparan sulfate biosynthetic genes into CHO cells. The heparan sulfate isolated from these cells was produced in protein and xeno-free conditions in processes that are amiable to good manufacturing practices at large scale. In addition, the inventors of the present disclosure have demonstrated that a targeted integration approach can generate CHO cell clones that produce heparan sulfate of consistent yield, composition, and bioactivity, streamlining the cell line development process. The inventors of the present disclosure believe that this technology can be used to generate recombinant heparan sulfate variants for a broad range of applications and would be more suitable for translation into commercial and clinical settings than heparan sulfate isolated from animal tissues.

DETAILED DESCRIPTION OF FIGURES

Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural, electrical and optical changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure. FIG. 1 shows an overview of RMCE targeted integration platform for heparan sulfate modifying enzymes and proteoglycan genes in CHO cells. A schematic representation of generation of stable cell pools via RMCE is shown. CHO-K1 cells were stably tagged with a Flp/FRT RMCE cassette (landing pad) via random integration. Cells bearing single landing pad was selected and used as master cell clone (MCL).

FIG. 2A shows a bar graph with the mass of heparan sulfate isolated per mL of CM from single gene expressing CHO lines at days 7 (white) and 14 (gray), with mean mass per mL media (mg) of two independent samples.

FIG. 2B shows stacked bar graphs with disaccharide compositional analysis of heparan sulfate variants isolated from singe gene expressing CHO CM at day 7 (i) or day 14 (ii), performed via enzymatic depolymerization and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean ± S.D. percentage composition of two independent samples analyzed in triplicate.

FIG. 2C shows a bar graph with SPR-based solution competition, and the percentage of FGF2 (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from single gene expressing CHO CM, with mean percentage FGF2 bound in solution ± S.D. of three independent experiments.

FIG. 2D shows a bar graph with MSCs cultured for 8 days ± heparan sulfate isolated from single gene expressing CHO CM (50 pg/mL; day 7 - white, day 14 - gray) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean normalized viable cell number ± S.D. of three independent experiments.

FIG. 2E shows a bar graph of the mass of heparan sulfate isolated per mL of CM at days 7 and 14 from dual gene expressing CHO CM, with mean mass per mL media (mg) of two independent samples.

FIG. 2F shows a stacked bar graph with a disaccharide compositional analysis of heparan sulfate variants performed via enzymatic depolymerization and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean ± S.D. percentage composition of two independent samples analyzed in triplicate. FIG. 2G shows a bar graph with SPR-based solution competition, and the percentage of FGF2 (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from dual gene expressing CHO CM, with mean percentage FGF2 bound in solution ± S.D. of three independent experiments.

FIG. 2H shows a bar graph with SPR-based solution competition, and the percentage of FGFRIa I lib Fc chimera (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from dual gene expressing CHO CM, with mean percentage FGFRIa I lib Fc chimera bound in solution ± S.D. of three independent experiments.

FIG. 21 shows a bar graph with MSCs cultured for 8 days ± heparan sulfate isolated from dual gene expressing CHO CM (50 pg/mL; day 7 - white, day 14 - gray) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean normalized viable cell number ± S.D. of three independent experiments.

FIG. 3A shows a bar graph with the mass of heparan sulfate isolated per mL of CM from HSPG expressing CHO lines at days 7 (white) and 14 (gray), with mean mass per mL media (mg) of two independent samples.

FIG. 3B shows a stacked bar graph with disaccharide compositional analysis of heparan sulfate variants performed via enzymatic depolymerization and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean ± S.D. percentage composition of two independent samples analyzed in triplicate.

FIG. 3C shows a bar graph with SPR-based solution competition, percentage of FGF2 (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from HSPG expressing CHO CM, mean percentage FGF2 bound in solution ± S.D. of three independent experiments.

FIG. 3D shows a bar graph with MSCs cultured for 8 days ± heparan sulfate isolated from HSPG expressing CHO CM (50 pg/mL; day 7 - white, day 14 - gray) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean normalized viable cell number ± S.D. of three independent experiments. FIG. 4A shows a bar graph of the mass of heparan sulfate isolated per mL of CM at days 7 and 14 from triple gene expressing CHO CM, with mean mass per mL media (mg) of two independent samples.

FIG. 4B shows a stacked bar graph of disaccharide compositional analysis of heparan sulfate variants performed via enzymatic depolymerization and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean percentage composition of two independent samples.

FIG. 4C shows a bar graph of SPR-based solution competition, percentage of FGF2 (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from triple gene expressing CHO CM, with mean percentage FGF2 bound in solution ± S.D. of three independent experiments.

FIG. 4D shows a bar graph of SPR-based solution competition, percentage of FGFRIa lllb Fc chimera (12.5 nM) bound to day 7 (white) or day 14 (gray) heparan sulfate variants (10 pg/mL) isolated from triple gene expressing CHO CM, mean percentage FGFRIa lllb Fc chimera bound in solution ± S.D. of three independent experiments.

FIG. 4E shows a bar graph of MSCs cultured for 8 days ± HS (50 pg/mL; day 7 - white, day 14 - gray) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, mean normalized viable cell number ± S.D. of three independent experiments.

FIG. 5A shows a bar graph with mass of heparan sulfate isolated per mL of CM from CHO K1 or HS-TG, with mean weight (mg) of two independent samples.

FIG. 5B shows a stacked bar graph with disaccharide compositional analysis of heparan sulfate variants performed via enzymatic depolymerisation and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean percentage composition of two independently digested samples.

FIG. 5C shows a line graph of a chromogenic factor Xa activity assay, with (i) absorbance measured at 405 nm with increasing concentrations of HS-TG (blue) or heparin (red), and (ii) anti-factor Xa activity in lU/mg based on the heparin USP standard. Datapoints represent mean absorbance at 405 nm from three independent experiments. FIG. 5D shows a graph of SPR, with representative sensorgrams (i-vi) of three independent experiments.

FIG. 5E shows differential scanning fluorimetry (i) melt curves generated by the denaturation of FGF2 ± increasing concentrations of HS-TG, and (ii) absolute melt temperature of FGF2 ± increasing concentrations of HS-TG, with representative melt curves (i) or mean absolute melt temperature (ii) of FGF2 ± HS8 or HS-TG from three independent experiments.

FIG. 5F shows blots and graphs with co-immunoprecipitation and Western blot detection of FGFRIa lllb and FGF2 complexes ± increasing concentrations (12.5, 25 and 50 pg/mL) of HS8 or HS-TG, with representative blots from three independent experiments.

FIG. 6A shows a bar graph of MSCs that were cultured for 8 days ± increasing concentrations of HS-TG (6.25, 12.5, 25, 50, and 100 pg/mL) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean ± S.D. of three independent experiments.

FIG. 6B shows a bar graph of MSCs that were cultured for 8 days ± HS-TG (50 pg/mL), FGF2 (1.25 ng/mL) or a combination of FGF2 and HS-TG with increasing concentrations of (i) IMBR1 (1 :2000, 1 :1000) or (ii) PD173074 (25, 50 or 100 nM) after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean ± S.D. of three independent experiments.

FIG. 6C shows a bar graph with mass of HS isolated per mL CM from 10 clones of the HS-TG cell line from a single HS preparation.

FIG. 6D shows a stacked bar graph with disaccharide compositional analysis of heparan sulfate variants performed via enzymatic depolymerization and SAX-HPLC, expressed as the relative percentage contribution of each disaccharide toward the total composition of the heparan sulfate chain, with mean ± S.D. of triplicate analysis from a single digest.

FIG. 6E shows a bar graph of MSCs that were cultured for 8 days ± HS-TG derived from 10 clones (50 pg/mL) or FGF2 (1.25 ng/mL), after which cell viability was measured using a Guava ViaCount. Data was then normalized to no treatment, with mean ± S.D. of three independent experiments. FIG. 7 shows gel images of junction PCR performed on gDNA of stably transfected pools to show correct cassette exchange for stable pools derived from RMCE. PCR product generated using 5’ integration primers confirmed integration across the FRT3 junction; and PCR produce generated using 3’ integration confirmed integration across the FRT junction.

FIG. 8 shows gel images of PCR bands of RT-PCR results of total RNA isolated from transfected cell lines. Via RMCE, 2 stably transfected cell pools that were generated for each of the constructs. Each pool expressed either a single heparan sulfate modifying enzyme, dual modifying enzyme or dual heparan sulfate modifying enzyme coupled with proteoglycan listed in Table 1. Control (Ctrl) pool are untransfected master cell line. The human HS modifying enzymes and proteoglycan gene expression in stable pools was confirmed by RT-PCR analysis.

FIG. 9A shows line graphs of FGF-2 solution competition (12.5 nM) with heparan sulfate isolated from single gene expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 9B shows line graphs of FGF-2 (12.5 nM) solution competition with heparan sulfate isolated from dual gene expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 9C shows line graphs of FGFR1 lllb Fc Chimera (12.5 nM) solution competition with heparan sulfate isolated from dual gene expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 9D shows line graphs of FGF-2 (12.5 nM) solution competition with heparan sulfate isolated from HSPG expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 9E shows line graphs of FGF-2 (12.5 nM) solution competition with heparan sulfate isolated from triple gene expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 9F shows line graphs of FGFR1 lllb Fc Chimera (12.5 nM) solution competition with heparan sulfate isolated from triple gene expressing CHO CM at day 7 (i) and day 14 (ii).

FIG. 10A shows a line graph of BMP2 (12.5 nM) solution competition with HS- TG (5, 10 and 20 pg/mL).

FIG. 10B shows a line graph of FGF2 (12.5 nM) solution competition with HS-TG (5, 10 and 20 pg/mL). FIG. 10C shows a line graph of FGFR1 lllb FC chimera (12.5 nM) solution competition with HS-TG (5, 10 and 20 pg/mL).

FIG. 10D shows a line graph of FGFR2 lllb FC chimera (12.5 nM) solution competition with HS-TG (5, 10 and 20 pg/mL).

FIG. 10E shows a line graph of PDGF-AB (12.5 nM) solution competition with HS-TG (5, 10 and 20 pg/mL).

FIG. 10F shows a line graph of PDGF-BB (12.5 nM) solution competition with HS-TG (5, 10 and 20 pg/mL).

FIG. 10G shows a line graph of SDF1a (100 nM) solution competition with HS- TG (5, 10 and 20 pg/mL).

FIG. 10H shows a line graph of TGFpi (100 nM) solution competition with HS- TG (5, 10 and 20 pg/mL).

FIG. 101 shows a line graph of VEGFies (12.5 nM) solution competition with HS- TG (5, 10 and 20 pg/mL).

FIG. 11 shows a schematic drawing of the constructs and gene combinations used in generating the CHO cell lines expressing the different heparan sulfate variants. The triangles refer to heterologous FRT sites, pA refers to polyadenylation signal.

FIG. 12 shows plots with HS-TG protein binding ELISA for FGF-2 (i), BMP-2 (ii), TGF 1 (iii), VEGF (iv), PDGF-AB (v), PDGF-BB (vi), IL-2 (vii) and IL-6 (viii).

FIG. 13 shows a bar graph of CHO triple gene heparan sulfate variants that enhance the stability of IL2. IL2 (0.1 ng/mL) was incubated ± CHO triple gene heparan sulfate variants (10 pg/mL) for 6 hours at 37°C, then used to stimulate the HEK-Blue IL2 reporter cell line for 24 h. Alkaline phosphatase activity was then measured by colourimetric assay and data normalised to IL2 activity with no incubation (0 h). APPLICATIONS

Embodiments of present disclosure disclosed herein provide a recombinant heparan sulfate.

Advantageously, the present disclosure provides a technology that can be applied to produce heparan sulfate that can optimally bind to other heparan sulfate binding proteins (such as VEGF, BMP, PDGF, IL) for cell therapy.

Even more advantageously, the present disclosure provides heparan sulfate additives for cell media formulations for human cell therapy manufacturing.

The present disclosure also provides heparan sulfate additives for media formulations for generating clean meats.

The present disclosure also provides heparan sulfate additives linked to biomaterials for use in regenerative medicine.

The present disclosure also provides heparan sulfate additives for direct use in drug-like form as injectables for the treatment of injury or disease or trauma.

The present disclosure also provides heparan sulfate additives for formulation into cosmetic products.

It will be appreciated by a person skilled in the art that other variations and/or modifications may be made to the embodiments disclosed herein without departing from the spirit or scope of the disclosure as broadly described. For example, in the description herein, features of different exemplary embodiments may be mixed, combined, interchanged, incorporated, adopted, modified, included etc. or the like across different exemplary embodiments. The present embodiments are, therefore, to be considered in all respects to be illustrative and not restrictive.