Stem Cell Clinic

Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy – Science Advances

Abstract

Enzyme replacement therapy, in which a functional copy of an enzyme is injected either systemically or directly into the brain of affected individuals, has proven to be an effective strategy for treating certain lysosomal storage diseases. The inefficient uptake of recombinant enzymes via the mannose-6-phosphate receptor, however, prohibits the broad utility of replacement therapy. Here, to improve the efficiency and efficacy of lysosomal enzyme uptake, we exploited the strategy used by diphtheria toxin to enter into the endolysosomal network of cells by creating a chimera between the receptor-binding fragment of diphtheria toxin and the lysosomal hydrolase TPP1. We show that chimeric TPP1 binds with high affinity to target cells and is efficiently delivered into lysosomes. Further, we show superior uptake of chimeric TPP1 over TPP1 alone in brain tissue following intracerebroventricular injection in mice lacking TPP1, demonstrating the potential of this strategy for enhancing lysosomal storage disease therapy.

Lysosomal storage diseases (LSDs) are a group of more than 70 inherited childhood diseases characterized by an accumulation of cellular metabolites arising from deficiencies in a specific protein, typically a lysosomal hydrolase. Although each individual disease is considered rare, LSDs have a combined incidence of between 1/5000 and 1/8000 live births, and together, they account for a substantial proportion of the neurodegenerative diseases in children (1). The particular age of onset for a given LSD varies depending on the affected protein and the percentage of enzymatic activity still present; however, in most cases, symptoms manifest early in life and progress insidiously, affecting multiple tissues and organs (2). In all but the mildest of cases, disease progression results in severe physical disability, possible intellectual disability, and a shortened life expectancy, with death occurring in late childhood or early adolescence.

As they are monogenic diseases, reintroducing a functional form of the defective enzyme into lysosomes is in principle a viable strategy for treating LSDs. Enzyme replacement therapy (ERT) is now approved for the treatment of seven LSDs, and clinical trials are ongoing for five others (3). However, delivering curative doses of recombinant lysosomal enzymes into lysosomes remains a major challenge in practice. ERT typically takes advantage of a specific N-glycan posttranslational modification, mannose-6-phosphorylation (M6P), which controls trafficking of endogenous lysosomal enzymes, as well as exogenous uptake of lysosomal enzymes from circulation by cells having the cation-independent M6P receptor (CIMPR) (4). Hence, a combination of factors including (i) the abundance of the M6P receptor in the liver, (ii) poor levels of CIMPR expression in several key target tissue types such as bone and skeletal muscle, (iii) incomplete and unpredictable M6P labeling of recombinant enzymes, and (iv) the highly variable affinity of recombinant lysosomal enzymes for CIMPR [viz., Kds (dissociation constants) ranging from low to mid micromolar (5, 6)] all contribute to diminishing the overall effectiveness of therapies using CIMPR for cell entry (3).

To improve the delivery of therapeutic lysosomal enzymes, we drew inspiration from bacterial toxins, which, as part of their mechanism, hijack specific host cellsurface receptors to gain entry into the endolysosomal pathway. While we and others have explored exploiting this pathway to deliver cargo into the cytosol (7, 8), here we asked whether this same approach could be used to enhance the delivery of lysosomal enzymes into lysosomes. We choose the diphtheria toxin (DT)diphtheria toxin receptor (DTR) system owing to the ubiquitous nature of the DTR, in particular its high expression levels on neurons.

Corynebacterium diphtheriae secretes DT exotoxin, which is spread to distant organs by the circulatory system, where it affects the lungs, heart, liver, kidneys, and the nervous system (9). It is estimated that 75% of individuals with acute disease also develop some form of peripheral or cranial neuropathy. This multiorgan targeting results from the fact that the DTR, heparin-binding EGF (epidermal growth factor)like growth factor (HBEGF), is ubiquitously expressed. The extent to which DT specifically targets difficult-to-access tissues such as muscle and bone, however, is not currently known.

DT is a three-domain protein that consists of an N-terminal ADP (adenosine diphosphate)ribosyl transferase enzyme (DTC), a central translocation domain (DTT), and a C-terminal receptorbinding domain (DTR). The latter is responsible for both binding cell surface HBEGF with high affinity [viz., Kd = 27 nM (10)] and triggering endocytosis into early endosomes (Fig. 1A). Within endosomes, DTT forms membrane-spanning pores that serve as conduits for DTC to enter the cytosol where it inactivates the host protein synthesis machinery. The remaining portions of the toxin remain in the endosomes and continue to lysosomes where they are degraded (11, 12). We hypothesized that the receptor-binding domain, lacking any means to escape endosomes, would proceed with any attached cargo to lysosomes and, thus, serve as a means to deliver cargo specifically into lysosomes following high-affinity binding to HBEGF.

(A) DT intoxication pathway (left), DT domain architecture, and LTM structure (right). (B and C) DTK51E/E148K, LTM, mCherry-LTM, and LTM-mCherry compete with wild-type DT for binding and inhibit its activity in a dose-dependent manner with IC50 (median inhibitory concentration) values of 46.9, 10.1, 52.7, and 76.1 nM, respectively (means SD; n = 3). (D and E) C-terminal and N-terminal fusions of LTM to mCherry were immunostained (red) and observed to colocalize with the lysosomal marker LAMP1 (39). (F) Fractional co-occurrence of the red channel with the green channel (Manders coefficient M2) were calculated for mCherry-LTM and LTM-mCherry and were found to be 0.61 0.10 and 0.52 0.11, respectively (means SD; n = 6).

In this study, we generated a series of chimeric proteins containing the DTR-binding domain, DTR, with the goal of demonstrating the feasibility of delivering therapeutic enzymes into lysosomes through the DT-HBEGF internalization pathway. We showed that DTR serves as a highly effective and versatile lysosome-targeting moiety (LTM). It can be placed at either the N or C terminus of the cargo, where it retains its high-affinity binding to HBEGF and the ability to promote trafficking into lysosomes both in vitro and in vivo. On the basis of its advantages, over M6P-mediated mechanisms, we further investigated the utility of LTM for the lysosomal delivery of human tripeptidyl peptidase-1 (TPP1) with the long-term goal of treating Batten disease.

To evaluate whether the DTR-binding fragment could function autonomously to traffic cargo into lysosomes, we first asked whether the isolated 17-kDa DTR fragment could be expressed independently from DT holotoxin and retain its affinity for HBEGF. We cloned, expressed, and purified the receptor-binding fragment and evaluated its ability to compete with full-length DT for the DTR, HBEGF. Before treating cells with a fixed dose of wild-type DT that completely inhibits protein synthesis, cells were incubated with a range of concentrations of LTM or a full-length, nontoxic mutant of DT (DTK51E/E148K). LTM-mediated inhibition of wild-type DT-mediated toxicity was equivalent to nontoxic DT (Fig. 1B), demonstrating that the receptor-binding fragment can be isolated from the holotoxin without affecting its ability to fold and bind cell surface HBEGF. Next, we evaluated whether LTM had a positional bias (i.e., was able to bind HBEGF with a fusion partner when positioned at either terminus). To this end, we generated N- and C-terminal fusions of LTM to the model fluorescent protein mCherry (i.e., mCherry-LTM and LTM-mCherry). To determine binding of each chimera to HBEGF, we quantified the ability of each chimera to compete with wild-type DT on cells in the intoxication assay. Both constructs competed with wild-type DT to the same extent as LTM alone and DTK51E/E148K (Fig. 1C), demonstrating that LTM is versatile and autonomously folds in different contexts.

To evaluate intracellular trafficking, HeLa cells were treated with either LTM-mCherry or mCherry-LTM and then fixed and stained 4 hours later with an antibody against the lysosomal marker LAMP1. In both cases, we observed significant uptake of the fusion protein (Fig. 1, D and E). We calculated Manders coefficients (M2) to quantify the extent to which signal in the red channel (LTM-mCherry and mCherry-LTM) was localizing with signal in the green channel (LAMP1). The fraction of red/green co-occurrence was calculated to be 0.61 for mCherry-LTM and 0.52 for LTM-mCherry, indicating trafficking to the lysosomal compartments of the cells and no significant difference (P = 0.196) between the two orientations of chimera (Fig. 1F). Together, these results confirm that the LTM is capable of binding HBEGF and trafficking associated cargo into cells and that the LTM can function in this manner at either terminus of a fusion construct.

With minimal positional bias observed in the mCherry fusion proteins, we next screened LTM fusions to TPP1 to identify a design that maximizes expression, stability, activity, and, ultimately, delivery. TPP1 is a 60-kDa lysosomal serine peptidase encoded by the CLN2 gene, implicated in neuronal ceroid lipofuscinosis type 2 or Batten disease. Loss of function results in the accumulation of lipofuscin, a proteinaceous, autofluorescent storage material (13). Exposure to the low-pH environment of the lysosome triggers autoproteolytic activation of TPP1 and release of a 20-kDa propeptide that occludes its active site. From a design perspective, we favored an orientation in which the LTM was N terminal to TPP1, as autoprocessing of TPP1 would result in the release of the upstream LTM-TPP1 propeptide, liberating active, mature TPP1 enzyme in the lysosome (Fig. 2A). Given the need for mammalian expression of lysosomal enzymes, we generated synthetic genetic fusions of the LTM to TPP1, in which we converted the codons from bacterially derived DT into the corresponding mammalian codons. Human embryonic kidney (HEK) 293F suspension cells stably expressing recombinant TPP1 (rTPP1) and TPP1 with an N-terminal LTM fusion (LTM-TPP1) were generated using the piggyBac transposon system (14). A C-terminal construct (TPP1-LTM) was also produced; however, expression of this chimera was poor in comparison with rTPP1 and LTM-TPP1 (~0.4 mg/liter, cf. 10 to 15 mg/liter).

(A) Design of LTM-TPP1 fusion protein and delivery schematic. (B) Enzyme kinetics of rTPP1 and LTM-TPP1 against the synthetic substrate AAF-AMC are indistinguishable. Michaelis-Menten plots were generated by varying [AAF-AMC] at a constant concentration of 10 nM enzyme (means SD; n = 3). Plots and kinetic parameters were calculated with GraphPad Prism 7.04. (C) Maturation of TPP1 is unaffected by the N-terminal fusion of LTM. (D) LTM-TPP1 inhibits wild-type DT activity in a dose-dependent manner (IC50 of 17.2 nM), while rTPP1 has no effect on protein synthesis inhibition by DT (means SD; n = 3). (E) LTM and DTR-TPP1 bind HBEGF with apparent Kds of 13.3 and 19.1 nM, respectively. (F) LTM-TPP1 (39) colocalizes with LAMP1 staining (red).

The activity of rTPP1 and LTM-TPP1 against the tripeptide substrate Ala-Ala-Phe-AMC (AAF-AMC) was assessed to determine any effects of the LTM on TPP1 activity. The enzyme activities of rTPP1 and LTM-TPP1 were determined to be equivalent, as evidenced through measurements of their catalytic efficiency (Fig. 2B), demonstrating that there is no inference by LTM on the peptidase activity of TPP1. Maturation of LTM-TPP1 through autocatalytic cleavage of the N-terminal propeptide was analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) (Fig. 2C). Complete processing of the zymogen at pH 3.5 and 37C occurred between 5 and 10 min, which is consistent with what has been observed for the native recombinant enzyme (15).

The ability of LTM-TPP1 to compete with DT for binding to extracellular HBEGF was first assessed with the protein synthesis competition assay. Similar to LTM, mCherry-LTM, and LTM-mCherry, LTM-TPP1 prevents protein synthesis inhibition by 10 pM DT with an IC50 (median inhibitory concentration) of 17.2 nM (Fig. 2D). As expected, rTPP1 alone was unable to inhibit DT-mediated entry and cytotoxicity. To further characterize this interaction, we measured the interaction between LTM and LTM-TPP1 and recombinant HBEGF using surface plasmon resonance (SPR) binding analysis (Fig. 2E). By SPR, LTM and LTM-TPP1 were calculated to have apparent Kds of 13.3 and 19.1 nM, respectively, values closely corresponding to the IC50 values obtained from the competition experiments (10.1 and 17.2 nM, respectively). Consistent with these results, LTM-TPP1 colocalizes with LAMP1 by immunofluorescence (Fig. 2F).

To study uptake of chimeric fusion proteins in cell culture, we generated a cell line deficient in TPP1 activity. A CRISPR RNA (crRNA) was designed to target the signal peptide region of TPP1 in exon 2 of CLN2. Human HeLa Kyoto cells were reverse transfected with a Cas9 ribonucleoprotein complex and then seeded at low density into a 10-cm dish. Single cells were expanded to colonies, which were picked and screened for TPP1 activity. A single clone deficient in TPP1 activity was isolated and expanded, which was determined to have ~4% TPP1 activity relative to wild-type HeLa Kyoto cells plated at the same density (Fig. 3A). The small residual activity observed is likely the result of another cellular enzyme processing the AAFAMC (7-amido-4-methlycoumarin) substrate used in this assay, as there is no apparent TPP1 protein being produced (Fig. 3B). Sanger sequencing of the individual alleles confirmed complete disruption of the CLN2 gene (fig. S1). In total, three unique mutations were identified within exon 2 of CLN2: a single base insertion resulting in a frameshift mutation and two deletions of 24 and 33 base pairs (bp), respectively.

(A) CLN2 knockout cells exhibit ~4% TPP1 activity relative to wild-type HeLa Kyoto cells (means SD; n = 3). (B) Western blotting against TPP1 reveals no detectable protein in the knockout cells. (C) (Left) In vitro maturation of pro-rTPP1 and LTM-TPP1 (16 ng) was analyzed by Western blot. (Right) TPP1 present in wild-type (WT) and TPP1/ cells, and TPP1/ cells treated with 100 nM rTPP1 and LTM-TPP1. (D) Uptake of rTPP1 and LTM-TPP1 into HeLa Kyoto TPP1/ cells was monitored by TPP1 activity (means SD; n = 4). (E) TPP1 activity present in HeLa Kyoto TPP1/ cells following a single treatment with 50 nM LTM-TPP1 (means SD; n = 3).

Next, we compared the delivery and activation of rTPP1 and LTM-TPP1 into lysosomes by treating TPP1/ cells with a fixed concentration of the enzymes (100 nM) and by analyzing entry and processing by Western blot (Fig. 3C). In both cases, most enzymes were present in the mature form, indicating successful delivery to the lysosome; however, the uptake of LTM-TPP1 greatly exceeded the uptake of rTPP1. As both rTPP1 and LTM-TPP1 receive the same M6P posttranslational modifications promoting their uptake by CIMPR, differences in their respective uptake should be directly attributable to uptake by HBEGF. To quantify the difference in uptake and lysosomal delivery, cells were treated overnight with varying amounts of each enzyme, washed, lysed, and assayed for TPP1 activity. The activity assays were performed without a preactivation step, so signal represents protein that has been activated in the lysosome. For both constructs, we observed a dose-dependent increase in delivery of TPP1 to the lysosome (Fig. 3D). Delivery of LTM-TPP1 was significantly enhanced compared with TPP1 alone at all doses, further demonstrating that uptake by HBEGF is more efficient than that by CIMPR alone. TPP1 activity in cells treated with LTM-TPP1 was consistently ~10 greater than that of cells treated with rTPP1, with the relative difference increasing at the highest concentrations tested. This may speak to differences in abundance, replenishment, and/or recycling of HBEGF versus CIMPR, in addition to differences in receptor-ligand affinity. Uptake of LTM-TPP1 and rTPP1 into several other cell types yielded similar results (fig. S2). To assess the lifetime of the delivered enzyme, cells were treated with LTM-TPP1 (50 nM) and incubated overnight. Cells were washed and incubated with fresh media, and TPP1 activity was assayed over the course of several days. Cells treated with LTM-TPP1 still retained measurable TPP1 activity at 1 week after treatment (Fig. 3E).

While the DT competition experiment demonstrated that HBEGF is involved in the uptake of LTM-TPP1 but not rTPP1 (Fig. 2D), it does not account for the contribution of CIMPR to uptake. Endoglycosidase H (EndoH) cleaves between the core N-acetylglucosamine residues of high-mannose N-linked glycans, leaving behind only the asparagine-linked N-acetylglucosamine moiety. Both rTPP1 and LTM-TPP1 were treated with EndoH to remove any M6P moieties, and delivery into Hela TPP1/ was subsequently assessed. While rTPP1 uptake is completely abrogated by treatment with EndoH, LTM-TPP1 uptake is only partially decreased (Fig. 4), indicating that while HBEGF-mediated endocytosis is the principal means by which LTM-TPP1 is taken up into cells, uptake via CIMPR still occurs. The fact that CIMPR uptake is still possible in the LTM-TPP1 fusion means that the fusion is targeted to two receptors simultaneously, increasing its total uptake and, potentially, its biodistribution.

Uptake of LTM-TPP1 via the combination of HBEGF and CIMPR was shown to be 3 to 20 more efficient than CIMPR alone in cellulo (fig. S2). To interrogate this effect in vivo, TPP1-deficient mice (TPP1tm1pLob or TPP1/) were obtained as a gift from P. Lobel at Rutgers University. Targeted disruption of the CLN2 gene was achieved by insertion of a neo cassette into intron 11 in combination with a point mutation (R446H), rendering these mice TPP1 null by both Western blot and enzyme activity assay (16). Prior studies have demonstrated that direct administration of rTPP1 into the cerebrospinal fluid (CSF) via intracerebroventricular or intrathecal injection results in amelioration of disease phenotype (17) and even extension of life span in the disease mouse (18). To compare the uptake of LTM-TPP1 and rTPP1 in vivo, the enzymes were injected into the left ventricle of 6-week-old TPP1/ mice. Mice were euthanized 24 hours after injection, and brain homogenates of wild-type littermates, untreated, and treated mice were assayed for TPP1 activity (Fig. 5A). Assays were performed without preactivation, and therefore, the results report on enzyme that has been taken up into cells, trafficked to the lysosome, and processed to the mature form.

(A) Assay schematic. (B) TPP1 activity in brain homogenates of 6-week-old mice injected with two doses (5 and 25 g) of either rTPP1 or LTM-TPP1 (5 g, P = 0.01; 25 g, P = 0.002). (C) TPP1 activity in brain homogenates following a single 25-g dose of LTM-TPP1, 1, 7, and 14 days postinjection. Data are presented as box and whisker plots, with whiskers representing minimum and maximum values from n 4 mice per group. Statistical significance was calculated using paired t tests with GraphPad Prism 7.04.

While both enzymes resulted in a dose-dependent increase in TPP1 activity, low (5 g) and high (25 g) doses of rTPP1 resulted in only modest increases of activity, representing ~6 and ~26% of the wild-type levels of activity, respectively (Fig. 5B). At the same doses, LTM-TPP1 restored ~31 and ~103% of the wild-type activity. To assess the lifetime of enzyme in the brain, mice were injected intracerebroventricularly with 25 g of LTM-TPP1 and euthanized either 1 or 2 weeks postinjection. Remarkably, at 1 week postinjection, ~68% of TPP1 activity was retained (compared with 1 day postinjection), and after 2 weeks, activity was reduced to ~31% (Fig. 5C).

ERT is a lifesaving therapy that is a principal method of treatment in non-neurological LSDs. Uptake of M6P-labeled enzymes by CIMPR is relatively ineffective due to variable receptor affinity (5, 6), heterogeneous expression of the receptor, and incomplete labeling of recombinantly produced enzymes (19). Despite its inefficiencies and high cost (~200,000 USD per patient per year) (20), it remains the standard of care for several LSDs, as alternative treatment modalities (substrate reduction therapy, gene therapy, and hematopoietic stem cell transplantation) are not effective, not as well developed, or inherently riskier (2125). Improving the efficiency and distribution of recombinant enzyme uptake may help address some of the current shortcomings in traditional ERT.

Several strategies have been used to increase the extent of M6P labeling on recombinantly produced lysosomal enzymes: engineering mammalian and yeast cell lines to produce more specific/uniform N-glycan modification (19, 26, 27), chemical or enzymatic modification of N-glycans posttranslationally (28), and covalent coupling of M6P (29). M6P-independent uptake of a lysosomal hydrolase by CIMPR has been demonstrated for both -glucuronidase (28) and acid -glucosidase (30, 31). In the latter work, a peptide tag (GILT) targeting insulin-like growth factor II receptor (IGF2R) was fused to recombinant alpha glucosidase, which enabled receptor-mediated entry into cells. CIMPR is a ~300-kDa, 15-domain membrane protein with 3 M6P-binding domains and 1 IGF2R domain. By targeting the IGF2R domain with a high-affinity (low nanomolar) peptide rather than the low-affinity M6P-binding domain, the authors were able to demonstrate a >20-fold increase in the uptake of a GAA-peptide fusion protein in cell culture and a ~5-fold increase in the ability to clear built-up muscle glycogen in GAA-deficient mice.

In this study, we have demonstrated efficient uptake and lysosomal trafficking of a model lysosomal enzyme, TPP1, via a CIMPR-independent route, using the receptor-binding domain of a bacterial toxin. HBEGF is a member of the EGF family of growth factors, and DT is its only known ligand. Notably, it plays roles in cardiac development, wound healing, muscle contraction, and neurogenesis; however, it does not act as a receptor in any of these physiological processes (32). Intracellular intoxication by DT is the only known process in which HBEGF acts as a receptor, making it an excellent candidate receptor for ERT, as there is no natural ligand with which to compete. Upon binding, DT is internalized via clathrin-mediated endocytosis and then trafficked toward lysosomes for degradation (33, 34). Acidification of endosomal vesicles by vacuolar ATPases (adenosine triphosphatases) promotes insertion of DTT into the endosomal membrane and subsequent translocation of the catalytic DTC domain into the cytosol. In the absence of an escape mechanism, the majority of internalized LTM should be trafficked to the lysosome, as we have demonstrated with our chimera (Figs. 2F and 3C). Uptake of LTM-TPP1 in vitro is robustly relative to rTPP1 (Fig. 3D and fig. S2), and TPP1 activity is sustained in the lysosome for a substantial length of time (Fig. 3E). We have also demonstrated that the increase in uptake efficiency that we observed in cell culture persists in vivo. TPP1 activity in the brains of CLN2-null mice was significantly greater in animals treated with intracerebroventricularly injected LTM-TPP1, as compared with those treated with TPP1 at two different doses (Fig. 5B), and, remarkably, this activity persists with an apparent half-life of ~8 days (Fig. 5C).

An important consideration for further development of the LTM platform for clinical development is the potential immunogenicity of using a bacterial fragment in this context. Previously, we demonstrated that the receptor-binding fragment of DT could be replaced with a human scFv (single-chain fragment variable) targeting HBEGF (8). With our demonstration of the potential for targeting HBEGF for LSDs, future efforts will focus on increasing the affinity and specificity of these first-generation humanized LTMs to develop high-affinity chimeras with greatly reduced immunogenicity for further development.

While the ability of LTM-TPP1 to affect disease progression has yet to be determined, recent positive clinical trial results (35) and the subsequent approval of rTPP1 (cerliponase alfa) for treatment of neuronal ceroid lipofuscinosis 2 (NCL2) provide support for this approach. In that clinical trial, 300 mg of rTPP1 was administered by biweekly intracerebroventricular injection to 24 affected children, and this was able to prevent disease progression. While this dose is of the same order of magnitude as other approved ERTs (<1 to 40 mg/kg) (36, 37), it represents a substantial dose, especially considering that it was delivered to a single organ. Improving the efficiency of uptake by targeting an additional receptor as we have done here, is expected to greatly decrease the dose required to improve symptoms, while at the same time decreasing costs and the chances of dose-dependent side effects.

DTK51E/E148K, LTM, LTM-mCherry, mCherry-LTM, and HBEGF constructs were cloned using the In-Fusion HD cloning kit (Clontech) into the Champion pET SUMO expression system (Invitrogen). Recombinant proteins were expressed as 6His-SUMO fusion proteins in Escherichia coli BL21(DE3)pLysS cells. Cultures were grown at 37C until an OD600 (optical density at 600 nm) of 0.5, induced with 1 mM IPTG (isopropyl--d-thiogalactopyranoside) for 4 hours at 25C. Cell pellets harvested by centrifugation were resuspended in lysis buffer [20 mM tris (pH 8.0), 160 mM NaCl, 10 mM imidazole, lysozyme, benzonase, and protease inhibitor cocktail] and lysed by three passages through an EmulsiFlex C3 microfluidizer (Avestin). Following clarification by centrifugation at 18,000g for 20 min and syringe filtration (0.2 m), soluble lysate was loaded over a 5-ml His-trap FF column (GE Healthcare) using an AKTA FPLC. Bound protein was washed and eluted over an imidazole gradient (20 to 150 mM). Fractions were assessed for purity by SDS-PAGE, pooled, concentrated, and frozen on dry ice in 25% glycerol for storage at 80C.

TPP1 cDNA was obtained from the SPARC BioCentre (The Hospital for Sick Children) and cloned into the piggyBac plasmid pB-T-PAF (J.M.R., University of Toronto) using Not I and Asc I restriction sites to generate two expression constructs (pB-T-PAF-ProteinA-TEV-LTM-TPP1 and pB-T-PAF-ProteinA-TEV-TPP1). Stably transformed expression cell lines (HEK293F) were then generated using the piggyBac transposon system, as described (14). Protein expression was induced with doxycycline, and secreted fusion protein was separated from expression media using immunoglobulin G (IgG) Sepharose 6 fast flow resin (GE Healthcare) in a 10-ml Poly-Prep chromatography column (Bio-Rad). Resin was washed with 50 column volumes of wash buffer [10 mM tris (pH 7.5) and 150 mM NaCl] and then incubated overnight at 4C with TEV (Tobacco Etch Virus) protease to release the recombinant enzyme from the Protein A tag. Purified protein was then concentrated and frozen on dry ice in 50% glycerol for storage at 80C.

Cellular intoxication by DT was measured using a nanoluciferase reporter strain of Vero cells (Vero NlucP), as described previously (8). Briefly, Vero NlucP cells were treated with a fixed dose of DT at EC99 (10 pM) and a serial dilution of LTM, LTM-mCherry, mCherry-LTM, DTK51E/E148K, LTM-TPP1, or rTPP1 and incubated overnight (17 hours) at 37C. Cell media was then replaced with a 1:1 mixture of fresh media and Nano-Glo luciferase reagent (Promega), and luminescence was measured using a SpectraMax M5e (Molecular Devices). Results were analyzed with GraphPad Prism 7.04.

SPR analysis was performed on a Biacore X100 system (GE Healthcare) using a CM5 sensor chip. Recombinant HBEGF was immobilized to the chip using standard amine coupling at a concentration of 25 g/ml in 10 mM sodium acetate (pH 6.0) with a final response of 1000 to 2500 resonance units (RU). LTM and LTM-TPP1 were diluted in running buffer [200 mM NaCl, 0.02% Tween 20, and 20 mM tris (pH 7.5)] at concentrations of 6.25 to 100 nM and injected in the multicycle analysis mode with a contact time of 180 s and a dissociation time of 600 s. The chip was regenerated between cycles with 10 mM glycine (pH 1.8). Experiments were performed in duplicate using two different chips. Binding data were analyzed with Biacore X100 Evaluation Software version 2.0.2, with apparent dissociation constants calculated using the 1:1 steady-state affinity model.

HeLa cells were incubated with LTM-mCherry (0.5 M), mCherry-LTM (0.5 M), or LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. mCherry constructs were visualized with a rabbit polyclonal antibody against mCherry (Abcam, ab16745) and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific). LAMP1 was stained with a mouse primary antibody (DSHB 1D4B) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific).

Colocalization was quantified using the Volocity (PerkinElmer) software package to measure Manders coefficients of mCherry signal with LAMP1 signal. The minimal threshold for the 488- and 568-nm channels was adjusted to correct the background signal. The same threshold for both channels was used for all the cells examined.

CLN2/ fibroblast 19494 were incubated with LTM-TPP1 (2 M) for 2 hours. Cells were washed with ice-cold PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100. LTM-TPP1 was visualized with a mouse monoclonal against TPP1 (Abcam, ab54685) and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific). LAMP1 was stained with rabbit anti-LAMP1 and anti-rabbit Alexa Fluor 568 (Thermo Fisher Scientific).

TPP1 protease activity was measured using the synthetic substrate AAF-AMC using a protocol adapted from Vines and Warburton (38). Briefly, enzyme was preactivated in 25 l of activation buffer [50 mM NaOAc (pH 3.5) and 100 mM NaCl] for 1 hour at 37C. Assay buffer [50 mM NaOAc (pH 5.0) and 100 mM NaCl] and substrate (200 M AAF-AMC) were then added to a final volume of 100 l. Fluorescence (380 nm excitation/460 nm emission) arising from the release of AMC was monitored in real time using a SpectraMax M5e (Molecular Devices). TPP1 activity in cellulo was measured similarly, without the activation step. Cells in a 96-well plate were incubated with 25 l of 0.5% Triton X-100 in PBS, which was then transferred to a black 96-well plate containing 75 l of assay buffer with substrate in each well.

crRNA targeting the signal peptide sequence in exon 2 of CLN2 was designed using the Integrated DNA Technologies (www.idtdna.com) design tool. The gRNA:Cas9 ribonucleoprotein complex was assembled according to the manufacturers protocol (Integrated DNA Technologies) and reverse transfected using Lipofectamine RNAiMAX (Thermo Fisher Scientific) into HeLa Kyoto cells (40,000 cells in a 96-well plate). Following 48 hours of incubation, 5000 cells were seeded into a 10-cm dish. Clonal colonies were picked after 14 days and transferred to a 96-well plate. Clones were screened for successful CLN2 knockout by assaying TPP1 activity and confirmed by Sanger sequencing and Western blot against TPP1 antibody (Abcam, ab54385).

The pro-form of TPP1 was matured in vitro to the active form in 50 mM NaOAc (pH 3.5) and 100 mM NaCl for 1 to 30 min at 37C. The autoactivation reaction was halted by the addition of 2 Laemmli SDS sample buffer containing 10% 2-mercaptoethanol and boiled for 5 min. Pro and mature TPP1 were separated by SDS-PAGE and imaged on a ChemiDoc gel imaging system (Bio-Rad).

Proteins or cellular lysate were separated by 4 to 20% gradient SDS-PAGE before being transferred to a nitrocellulose membrane using the iBlot (Invitrogen) dry transfer system. Membranes were then blocked for 1 hour with a 5% milktris-buffered saline (TBS) solution and incubated overnight at room temperature with a 1:100 dilution of mouse monoclonal antibody against TPP1 (Abcam, ab54685) in 5% milk-TBS. Membranes were washed 3 5 min with 0.1% Tween 20 (Sigma-Aldrich) in TBS before a 1-hour incubation with a 1:5000 dilution of sheep anti-mouse IgG horseradish peroxidase secondary antibody (GE Healthcare) in 5% milk-TBS. Chemiluminescent signal was developed with Clarity Western ECL substrate (Bio-Rad) and visualized on a ChemiDoc gel imaging system (Bio-Rad).

rTTP1 and LTM-TPP1 were treated with EndoH (New England Biolabs) to remove N-glycan modifications. Enzymes were incubated at 1 mg/ml with 2500 U of EndoH for 48 hours at room temperature in 20 mM tris (pH 8.0) and 150 mM NaCl in a total reaction volume of 20 l. Cleavage of N-glycans was assessed by SDS-PAGE, and concentrations were normalized to native enzyme-specific activities.

Cryopreserved TPP1+/ embryos were obtained from P. Lobel at Rutgers University and rederived in a C57/BL6 background at The Centre for Phenogenomics in Toronto. Animal maintenance and all procedures were approved by The Centre for Phenogenomics Animal Care Committee and are in compliance with the CCAC (Canadian Council on Animal Care) guidelines and the OMAFRA (Ontario Ministry of Agriculture, Food, and Rural Affairs) Animals for Research Act.

TPP1/ mice (60 days old) were anesthetized with isoflurane (inhaled) and injected subcutaneously with sterile saline (1 ml) and meloxicam (2 mg/kg). Mice were secured to a stereotactic system, a small area of the head was shaved, and a single incision was made to expose the skull. A high-speed burr was used to drill a hole at stereotaxic coordinates: anteroposterior (A/P), 1.0 mm; mediolateral (M/L), 0.3 mm; and dorsoventral (D/V), 3.0 mm relative to the bregma, and a 33-gauge needle attached to a 10-l Hamilton syringe was used to perform the intracerebroventricular injection into the left ventricle. Animals received either 1 or 5 l of enzyme (5 g/l), injected at a constant rate. Isoflurane-anesthetized animals were euthanized by transcardial perfusion with PBS. Brains were harvested and frozen immediately, then thawed and homogenized in lysis buffer [500 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and 50 mM Tris (pH 8.0)] using 5-mm stainless steel beads in TissueLyser II (Qiagen). In vitro TPP1 assay was performed, as described, minus the activation step.

Acknowledgments: We thank P. Lobel at Rutgers University for providing the TPP1-deficient mice. Funding: We are grateful to the Canadian Institutes of Health Research for funding. Author contributions: S.N.S.-M. devised and performed experiments and drafted the initial manuscript. G.L.B. provided materials and assisted in conceptualization and experimental design. X.Z., D.Z., and R.H. contributed to the experimental design and performed experiments. P.K.K. and B.A.M. contributed to the experimental design. J.M.R. contributed to the experimental design and revised the manuscript. R.A.M. assisted in conceptualization, contributed to the experimental design, and assisted in writing the manuscript. Competing interests: B.A.M. is a chief medical advisor at Taysha Gene Therapies. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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Exploiting the diphtheria toxin internalization receptor enhances delivery of proteins to lysosomes for enzyme replacement therapy - Science Advances

Haywards Heath woman’s bid to fund stem cell treatment to combat MS – Mid Sussex Times

Joceline Colvert was diagnosed with relapsing remitting Multiple Sclerosis in her early 20s and says she spent the first eight years researching and managing her condition while trying to mention it as little as possible to others and completing her Sound Production degree.

I spent most of my late 20s and early 30s finding ways to manage relapses, the symptoms of which have included whole body numbness, loss of the use of both hands, right eye blindness, vertigo and double vision, she said. Thankfully these symptoms did resolve however left scarring on my nerves. This results in reduced vision in my formerly blind eye and hands that dont function very well with repetitive tasks.

This semi-denial worked for me until about 2010 when I started to become a bit limpy which I did my best to hide. After a couple of memorable falls and fractures I decided to face up to being slightly rickety and got a hiking pole that I used occasionally in public. Since then Ive needed to get used to being visibly disabled, and switch between two hiking poles for very short distances and a wheelchair everywhere else.

Joceline, who lives with her husband and her five beloved cats and dogs, says she is not eligible for Haematopoietic Stem Cell Transplantation (HSCT), on the NHS which is the first treatment I have ever got excited about and believe could work. It could be truly life-changing.

As a result she is trying to raise money to fund the treatment herself.

HSCT is a procedure that aims to reset the faulty immune system which, in my case, is attacking my nervous system from within, Joceline said. Stem cells will be taken from my bone marrow or blood before my immune system is wiped out with chemotherapy. My cells are then reintroduced into my blood, where they grow a new immune system which will hopefully no longer attack my nerves or have any memory of MS.

The aim of HSCT is to completely halt progression, putting MS into remission with no requirement for immunosuppressant drug therapy. The success rate for relapsing remitting MS is 80% - 90% which is absolutely phenomenal compared to the limited available drug treatments, which only aim to slow down disability.

HSCT is available on the NHS, however there is a very strict criteria for which I do not qualify. The expense of the treatment and the increased pressures on the public purse mean the NHS will only treat patients who have been diagnosed for fewer than 15 years.

I have been diagnosed for 18 years.

I had prepared myself for this possibility and, for the last year, have been researching treatment with The National Pirogov Medical Centre Russia (Moscow). Russia has been pioneering in their use of HSCT to treat MS and are world renowned for their expertise and care. Im excited to have a treatment date in March 2021 which fills me with hope for a future free from progression. I need your help to get there.

Joceline, who loves making stop-motion animation puppets and props and playing musical instruments, says the treatment will cost 40,800, and the flights 800.

She has launched a Go Fund Me page at https://gf.me/u/y538k2 which has already seen donations of more than 26,000.

I am incredibly grateful for any help you can give towards enabling me to access this life-changing treatment, she said.

After almost two decades of managing MS flare-ups and their consequences, its hard to put into words just what a future without them would mean to me.

Thank you for reading this and for any help you can put towards this goal.

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Haywards Heath woman's bid to fund stem cell treatment to combat MS - Mid Sussex Times

Family of sick girl whose stem cell donor pulled out at the last minute find a replacement – The Sun

THE campaigning family of a sick girl whose stem cell transplant match pulled out at the last minute have found a new one.

Evie Hodgson, eight, has a deadly blood disorder and was preparing for the operation when her donor cancelled without explanation.

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Her family launched an appeal to find another, telling their story on ITVs This Morning, which saw 25,000 people sign up and found a second match.

Evies mum Tina, 37, said: This is the best Christmas present we could ever wish for.

Evie, of Whitby, North Yorks, was diagnosed with aplastic anaemia in May.

Medics were delighted when their global search for a bone marrow donor found a match only for disaster to strike.

RAF worker Tina, married to chief executive Andy, 49, said: We got the call about the new match while Evie was being treated for an infection.

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"She said, Mummy, youre my hero. It was so emotional.

Evie, who has a brother, is to have the op at the Great North Childrens Hospital in Newcastle in January.

She said: Thank you so much to everyone. Youve saved my life.

GOT a story? RING The Sun on 0207 782 4104 or WHATSAPP on 07423720250 or EMAILexclusive@the-sun.co.uk

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Family of sick girl whose stem cell donor pulled out at the last minute find a replacement - The Sun

Bid to fund stem cell treatment in Mexico for woman with MS – Bournemouth Echo

A FORMER hairdresser from Bournemouth is appealing for people to help raise money to have life-saving surgery in Mexico to get rid of her Multiple Sclerosis once and for all.

Having been admitted to Royal Bournemouth Hospital for a suspected stroke or brain tumour in March 2017, at the age of 47, Kirsten Hannibal was found to have multiple lesions on her brain and was diagnosed with CIS which later progressed to MS.

During lockdown, Kirsten has researched into different ways to stop Multiple Sclerosis dead in its tracks, one of them being Hematopoietic Stem Cell Transplantation.

Although the procedure, which involves the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, is not widely accessible in the UK, it is available in Mexico, considered a world class hub for HSCT.

However she must raise over 40,000 to cover flights to Mexico as well as the cost of the procedure.

Vicky Dixon has set up a crowdfunding page to raise money for Kirstens medical procedure.

In a statement written on her crowdfunding page, she said: Our family are joining forces to raise the money needed to send our Kirsten to Mexico for Hematopoietic Stem Cell Transplantation treatment that is not universally available on the NHS, but will hopefully give Kirsten a chance of a future; a life free of pain, disability and heart breaking challenges.

We hope that Kirsten can follow the footsteps of other British MS sufferers and go to Mexico, a world class centre for HSCT, and cheaper than the UK, at the cost of 43,500.

The first large, randomised control trial, and several meta-analyses of HSCT, have confirmed that HSCT is a very effective therapy. This is now tipping the scales for HSCT becoming a mainstream treatment for MS in Britain.

However, the treatment has to take place before the MS becomes too advanced, and as it will be years before HSCT might be offered more widely, Kirsten would by then be swallowed up by the MS and not a suitable candidate for treatment.

Kirsten is on the brink of becoming too disabled for this treatment, hence the urgency of our appeal.

Sadly, the 46-year-old is now travelling a path similar to one her family have walked before.

In 1984 her mother at the age of 32 was diagnosed with lymphoblastic leukaemia and the Echo covered the story.

Her mother underwent aggressive chemotherapy and was the receiver of a ground-breaking treatment with a bone marrow transplant.

She was the first patient to receive this treatment in the south and, whilst the treatment was deemed a success, sadly her mother died.

Lynda Smiths legacy lives on because her bravery in allowing this treatment to take place is now the lifeline to many children and adults alike who survive leukaemia.

The treatment Kirsten is looking to have is similar to her mothers treatment, except it would be her own bone marrow that would be harvested. She will then be given chemotherapy and then the day Kirsten longs for, freedom from the disease.

The new birthday she dreams of is a stem cell birthday celebrated when the bone marrow is put back into her body giving her the chance of stopping Multiple Sclerosis.

So far, Kirstens fundraising appeal has raised 4,535, just over 10 per cent of her target.

To donate, visit https://www.gofundme.com/f/multiple-sclerosis-and-an-urgent-bid-for-freedom?utm_source=customer&utm_medium=email&utm_campaign=p_cp+sharesheet.

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Bid to fund stem cell treatment in Mexico for woman with MS - Bournemouth Echo

CollPlant to Supply rhCollagen to STEMCELL Technologies for Use in a Broad Range of Cell Culture Applications – PRNewswire

REHOVOT, Israel and VANCOUVER, BC, Dec. 10, 2020 /PRNewswire/ -- CollPlant (NASDAQ: CLGN), a regenerative medicine company, and STEMCELL Technologies, Canada's largest privately owned biotechnology company, which develops cell culture media, cell separation systems, instruments, and other reagents for life sciences research, today jointly announced they have entered into aproduct manufacturing and supply agreement. CollPlant will sell its proprietary recombinant human Type I collagen (rhCollagen), the world's first plant-based rhCollagen, to STEMCELL Technologies, which will incorporate CollPlant's product into cell culture media kits.

The recently signed agreement follows the companies' established business relationship, which started in 2014 when STEMCELL began purchasing and incorporating CollPlant's rhCollagen into some of its cell culture expansion and differentiation media kits. To date, hundreds of companies, as well as research and academic institutes, have used these kits for research and development projects. STEMCELL will distribute the kits globally for use in the regenerative medicine research market.

"Incorporation of rhCollagen into STEMCELL's cell culture applications sold to researchers worldwide is designed to help advance the science in a broad range of dynamic fields including stem cells, immunology, cancer, regenerative medicine, and cellular therapy. We are happy to have entered into this agreement with STEMCELL, which, as Canada's largest biotechnology company, is very well positioned to make rhCollagen-containing cell culture kits widely available in the market," stated Yehiel Tal, Chief Executive Officer of CollPlant. "The cell culture market is just one example of the vast potential of our rhCollagen platform technology in life science applications. We continuously evaluate new fields in which CollPlant's products and technologies have the potential to enable breakthroughs that improve patients' lives."

Dr. Sharon Louis, STEMCELL's Senior Vice President of Research and Development noted that "STEMCELL is pleased to utilize CollPlant's animal component free rhCollagen to promote cell attachment in several products that support the culture of diverse human progenitor cell types. The quality and animal component-free composition of CollPlant's rhCollagen is what first brought this product to STEMCELL's attention, and the robust performance rhCollagen provides with a variety of STEMCELL media is what we want to be able to provide to our customers. Upon entering into this agreement, STEMCELL and CollPlant will together provide high-quality reagents that will be used to further our understanding in life sciences and potentiate regenerative medicine research."

About STEMCELL Technologies

STEMCELL Technologies is Canada's largest biotechnology company. Based in Vancouver, STEMCELL supports life sciences research around the world with more than 2,500 specialized reagents, tools, and services. STEMCELL offers high-quality cell culture media, cell separation technologies, instruments, accessory products, and educational resources that are used by scientists advancing the stem cell, immunology, cancer, regenerative medicine, microbiology, and cellular therapy fields.

Find more information at http://www.stemcell.com

About CollPlant Biotechnologies

CollPlant is a regenerative and aesthetic medicine company focused on 3D bioprinting of tissues and organs, and medical aesthetics. Our products are based on our rhCollagen (recombinant human collagen) that is produced with CollPlant's proprietary plant based genetic engineering technology.

Our products address indications for the diverse fields of tissue repair, aesthetics and organ manufacturing, and, we believe, are ushering in a new era in regenerative and aesthetic medicine.

Our flagship rhCollagen BioInk product line is ideal for 3D bioprinting of tissues and organs. In October 2018, we entered into a licensing agreement with United Therapeutics, whereby United Therapeutics is using CollPlant's BioInks in the manufacture of 3D bioprinted lungs for transplant in humans.Recently, the parties announced the expansion of the collaboration with the exercise by United Therapeutics of its option to cover a second lifesaving organ, human kidneys.

Safe Harbor for Forward-Looking Statements

This press release may include forward-looking statements. Forward-looking statements may include, but are not limited to, statements relating to CollPlant's objectives, plans and strategies, as well as statements, other than historical facts, that address activities, events or developments that CollPlant intends, expects, projects, believes or anticipates will or may occur in the future. These statements are often characterized by terminology such as "believes," "hopes," "may," "anticipates," "should," "intends," "plans," "will," "expects," "estimates," "projects," "positioned," "strategy" and similar expressions and are based on assumptions and assessments made in light of management's experience and perception of historical trends, current conditions, expected future developments and other factors believed to be appropriate. Forward-looking statements are not guarantees of future performance and are subject to risks and uncertainties that could cause actual results to differ materially from those expressed or implied in such statements. Many factors could cause CollPlant's actual activities or results to differ materially from the activities and results anticipated in forward-looking statements, including, but not limited to, the following: the CollPlant's history of significant losses and its need to raise additional capital and its inability to obtain additional capital on acceptable terms, or at all; CollPlant's expectations regarding the timing and cost of commencing clinical trials; regulatory action with respect to rhCollagen-based products, including but not limited to acceptance of an application for marketing authorization, review and approval of such application, and, if approved, the scope of the approved indication and labeling; commercial success and market acceptance of the CollPlant's rhCollagen-based BioInk; CollPlant's ability to establish sales and marketing capabilities or enter into agreements with third parties and its reliance on third-party distributors and resellers; CollPlant's reliance on third parties to conduct some aspects of its product manufacturing; the scope of protection CollPlant is able to establish and maintain for intellectual property rights and the company's ability to operate its business without infringing the intellectual property rights of others; the overall global economic environment; the impact of competition and new technologies; general market, political, and economic conditions in the countries in which the company operates; projected capital expenditures and liquidity; changes in the company's strategy; and litigation and regulatory proceedings. More detailed information about the risks and uncertainties affecting CollPlant is contained under the heading "Risk Factors" included in CollPlant's most recent annual report on Form 20-F, filed with the SEC, and in other filings that CollPlant has made. The forward-looking statements contained in this press release are made as of the date of this press release and reflect CollPlant's current views with respect to future events, and CollPlant does not undertake, and specifically disclaims, any obligation to update or revise any forward-looking statements, whether as a result of new information, future events or otherwise.

Contact atCollPlant:

Eran Rotem Deputy CEO & CFO Tel: + 972-73-2325600 [emailprotected]

Contact at STEMCELL: Luba Metlitskaia Vice President, Business Development & Licensing [emailprotected]

SOURCE CollPlant

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CollPlant to Supply rhCollagen to STEMCELL Technologies for Use in a Broad Range of Cell Culture Applications - PRNewswire

NASA-partnered Pluristem crashes to Earth as it axes leading therapy – FierceBiotech

Israeli biotech Pluristem is canning its experimental phase 3 critical limb ischemia therapy after an outside review said it was no good.

Haifa, Israel-based Pluristems R&D operation is built upon placenta-derived adherent stromal cells, which the biotech has designed for use in patients of all human leukocyte antigen types. This approach is made possible by the low immunogenicity of the cells. Once inside the body, Pluristem hopes the cells will drive the healing of injured tissue.

But one of its leading contenders using this approach has been judged a failure in phase 3: An independent data monitoring committee (DMC) took a look at the ongoing data for its pivotal phase 3 in patients with critical limb ischemia (CLI), a severe obstruction of the arteries which markedly reduces blood flow to the extremities and can lead to amputation.

During this webinar, attendees will learn about Thermo Fisher Scientifics new 5KL bioreactor and how it benefits clients who outsource late-phase biologics drug substance manufacturing. An introduction of the 5KL bioreactor will be provided, as well as application data around performance and scalability, process economy comparison with traditional stainless steel bioreactors, and decision criteria that could be helpful in choosing between different cell culture strategies. Register Today!

The DMC said the test was unlikely to meet the primary endpoint, and that the CLI study population has experienced a substantial low number of events (major amputation of the index leg or death), different from what is known in clinical medicine for the rate of these events in this patient population. The lower than anticipated event rate in the placebo group reduced the statistical power of the study to meet its primary endpoint.

The biotech is now tossing out the therapy and will instead focus on other pipeline areas, including a long-shot stem cell attempt at treating COVID-19. The biotechs shares fell nearly 40% on the news.

We are deeply disappointed by the outcome of the CLI interim analysis. In light of the DMCs recommendation, we decided that it would be in the best interests of the company and its shareholders to terminate the CLI study and focus our resources and efforts on our other lead indications, said Pluristem CEO and President Yaky Yanay.

We expect to present topline clinical results during calendar year 2021, including our phase 3 study in muscle regeneration following hip fracture, phase 2 studies in Acute Respiratory Distress Syndrome associated with COVID-19 and our phase 1 study in incomplete hematopoietic recovery following hematopoietic cell transplantation. Pluristem is well positioned to advance and support future development of these indications.

Last year, Pluristem penned a deal with NASA to assess its cell therapies against the health problems caused by spending time in space, teaming up with NASAs Ames Research Center for the project, which focuses on using its PLX placenta-derived cell therapies to try to prevent or treat medical conditions that can occur during and after space missions, including conditions that affect the blood, bone, muscle, brain and heart.

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NASA-partnered Pluristem crashes to Earth as it axes leading therapy - FierceBiotech

Global Animal Stem Cell Therapy Market To Reach A New Threshold of Growth By 2026 – The Courier

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Global Animal Stem Cell Therapy Market To Reach A New Threshold of Growth By 2026 - The Courier

Cryo-shocked cancer cells for targeted drug delivery and vaccination – Science Advances

Abstract

Live cells have been vastly engineered into drug delivery vehicles to leverage their targeting capability and cargo release behavior. Here, we describe a simple method to obtain therapeutics-containing dead cells by shocking live cancer cells in liquid nitrogen to eliminate pathogenicity while preserving their major structure and chemotaxis toward the lesion site. In an acute myeloid leukemia (AML) mouse model, we demonstrated that the liquid nitrogentreated AML cells (LNT cells) can augment targeted delivery of doxorubicin (DOX) toward the bone marrow. Moreover, LNT cells serve as a cancer vaccine and promote antitumor immune responses that prolong the survival of tumor-bearing mice. Preimmunization with LNT cells along with an adjuvant also protected healthy mice from AML cell challenge.

Acute myeloid leukemia (AML) is a hematological malignancy with a dismal prognosis and 5-year survival of only 30% (1, 2). The standard-of-care cytoreductive chemotherapy induces AML remission (35), but disease relapse frequently occurs (6, 7). Hematopoietic stem cell transplantation (HSCT) in patients who achieve remission after chemotherapy represents the only curative approach so far (8, 9). However, HSCT is associated with either the lack of suitable hematopoietic stem cell donors or the high risk of transplantation-related mortality (10). Hence, there is an urgent need to find further strategies for AML treatment.

AML originates in the bone marrow, and bone marrow creates leukemia-niches that promote leukemia survival (11). Furthermore, biodistribution of chemotherapeutics to the bone marrow is frequently poor (12, 13), and higher doses of chemotherapy required to ablate leukemia are toxic to normal tissues. Thus, developing targeting drug delivery to the bone marrow may not only enhance the therapeutic index of chemotherapy but also reduce its toxicity to nonhematopoietic tissues. Nevertheless, it is still challenging to engineer bone marrowtargeting moieties and bypass the bloodbone marrow barriers (14). Leveraging cells intrinsic properties offers solutions to overcome these limitations (1517). Because AML cells naturally exhibit bone marrow homing capabilities (1820), we developed an approach to use AML cells as drug carriers while eliminating their intrinsic pathogenicity.

Here, we used a liquid nitrogenbased cryo-shocking method to obtain therapeutic dead cells. These cells maintained the intact structure allowing for drug encapsulation, but lost their proliferation ability and pathogenicity. Specifically, cryo-shocked AML cells kept their bone marrow homing capability and served as a drug delivery vehicle of doxorubicin (DOX), which is a critical drug used in the induction chemotherapy in AML. Cryo-shocked AML cells stimulated an immune response that was in conjunction with chemotherapy to eradicate leukemia in tumor-bearing mice. Preimmunization with LNT cells together with an adjuvant protected healthy mice from AML cell challenge. We thus proposed a dead cellbased delivery vehicle that can be rapidly manufactured for clinical use compared with other live cellbased drug delivery systems (21).

To obtain the liquid nitrogentreated (LNT) cells, AML cells were suspended in the cell cryopreservation medium and immersed in liquid nitrogen for 12 hours. LNT cells were then thawed at 37C and washed with phosphate-buffered saline (PBS) (Fig. 1A). When analyzed by confocal microscopy, LNT cells showed the same cellular structure as untreated live cells when assessed by nucleus and cytoskeleton staining (Fig. 1B). A slight decrease in cellular size was observed (Fig. 1C), with an average size of 11 m for LNT cells and 12 m for untreated live cells. The forward scatter (FSC) values measured by flow cytometry corroborated the cell size reduction of LNT cells, and similar side scatter (SSC) values suggested that the internal structure of LNT cells was maintained (Fig. 1D). Scanning electron microscopy (SEM) images revealed the sphere-like structure of LNT cells and the rougher cellular surface as compared with control live cells (Fig. 1E and fig. S1).

(A) Schematic of the procedure to prepare LNT cells. (B) Cellular structure of live and LNT C1498 cells. Cell nucleus was stained by Hoechst 33342 (blue), and cytoplasm F-actin was stained by AF488 phalloidin (green). Scale bars, 10 m. (C) Cellular sizes of live and LNT C1498 cells. The cells were imaged by confocal microscopy, and cellular size was measured by the software Nano Measurer (cell numbers = 200). (D) Flow cytometry analysis of live and LNT C1498 cells under same voltages. FSC, forward scatter; SSC, side scatter. (E) SEM images of live and LNT cells. Scale bars, 1 m. (F) Cell viability analysis of live and LNT cells by LIVE/DEAD viability kit. Calcein AM: live cells; EthD-1: dead cells. Scale bar, 10 m. (G) Cell viability analysis of live and LNT cells by CCK8 assay (n = 6). a.u., arbitrary unit. (H) In vivo proliferation of 2 106 luciferase tagged live and LNT C1498 cells indicated by the bioluminescence signal (n = 5). (I) Survival of mice after challenge with 2 106 live and LNT tumor cells (n = 5). Typical flow cytometry images (J) and DsRed intensities (K) of peripheral blood 20 days after challenge with live and LNT DsRed tagged C1498 cells (n = 3). MFI, mean fluorescence intensity. Data are presented as means SD (G and K). Statistical significance was calculated via the log-rank (Mantel-Cox) test (I) and ordinary one-way analysis of variance (ANOVA) (K). *P < 0.05, **P < 0.01. NS, not significant.

Next, we evaluated the cell viability of LNT cells. As shown in Fig. 1F, nearly all the LNT cells were labeled with EthD-1 (indicating dead cells) and did not show intact fluorescence signal of calcein AM (indicating live cells). In addition, LNT cells did not show proliferative activity as compared with live cancer cells as measured with cell counting kit-8 (CCK8) assay (Fig. 1G). Furthermore, we confirmed the necrosis-dependent cell death of LNT cells by annexin-Vpropidium iodide (PI) staining (fig. S2). We further verified the absence of pathogenicity of LNT cells in vivo. As shown in Fig. 1H, live C1498 AML cells quickly proliferated in mice and caused 100% death in 31 days, while mice receiving C1498 LNT cells exhibited no detectable bioluminescence signal, and all mice survived for at least 180 days (Fig. 1, H and I). Moreover, we quantitatively analyzed cancer cells in the peripheral blood at day 20 after injection. A notably higher DsRed signal was observed in mice injected with live C1498 cells, indicating a high portion of leukemia cells circulating in the blood, while the DsRed intensity for the mice challenged with LNT cells was similar to that of healthy mice (Fig. 1, J and K).

Leukemia cells exhibit bone marrow homing and resident capabilities, which are at least in part associated with the expression of CXCR4 and CD44 chemokine, two typical adhesion receptors that interact with bone marrow (18, 22, 23). SDSpolyacrylamide gel electrophoresis (PAGE) showed that most of the proteins expressed by live C1498 cells were retained in LNT cells (fig. S3A). CXCR4 and CD44 were detected in both live and LNT cells as assessed by confocal imaging and flow cytometry (Fig. 2, A and B, and fig. S3, B and C). Despite some reduction in expression levels, Western blotting analysis indicated that CXCR4 and CD44 expression were 39 and 60%, respectively, in LNT cells compared with live cells (fig. S3, D and E). The bone marrow homing capacity of LNT cells was also evaluated. Upon intravenous infusion, LNT cells exhibited similar accumulation efficiency in bone barrow compared with live C1498 cells (Fig. 2, C and D, and fig. S4A). Cell signal was notably higher compared with paraformaldehyde-fixed cells, which reflects the loss of bioactivities upon paraformaldehyde fixation (Fig. 2, C and D). LNT cells also distributed in the liver, kidney, and spleen (fig. S4B), and were cleared from the bloodstream within 24 hours (fig. S5).

CXCR4 (A) and CD44 (B) expression of live and LNT C1498 cells analyzed by confocal microscopy (top) and flow cytometry (bottom). Scale bars, 10 m. (C) Fluorescence images of bone isolated 6 hours after injection of cy5.5-labeled live C1498 cells, LNT C1498 cells, and paraformaldehyde-fixed C1498 cells. (D) Fluorescence intensities of the bone of indicated groups (n = 6). (E) Typical confocal image of DOX-loaded LNT cells. Scale bar, 10 m. (F) Cumulative release profile of DOX from LNT cell/DOX (n = 3). (G) Plasma DOX concentration after intravenous injection of free DOX and LNT cell/DOX with DOX dose of 2.5 mg/kg (n = 4). (H) Bone marrow DOX content 3 hours after administration of the drug (n = 3). Data are presented as means SD (D and F to H). Statistical significance was calculated via ordinary one-way ANOVA (D) and Students t test (G and H). *P < 0.05, **P < 0.01, ***P < 0.001.

Because nuclear and cytoplasmic cellular structures are preserved in LNT cells (Fig. 1B), we assessed if these cells can be payload with DOX, via DNA intercalation and the electrostatic interactions between DOX and cytoplasm proteins (2426), and deliver DOX to bone marrow. Briefly, DOX could be loaded into LNT cells via mixing and incubation with a loading capacity of 65 16 g per 1 107 LNT cells (Fig. 2E and fig. S6A). DOX was released from the drug-loaded LNT cells (LNT cell/DOX) in a sustained manner, and 81% of DOX was released within 10 hours (Fig. 2F). We then studied the in vitro cytotoxicity against C1498 cells of free DOX and LNT cell/DOX. The IC50 (median inhibitory concentration) values were 0.32 and 1.05 g/ml, respectively (fig. S6B). Even though free DOX exhibited higher cytotoxicity against C1498 cells in vitro, LNT cell/DOX allowed longer detection of DOX in the blood and higher DOX accumulation within the bone marrow (Fig. 2, G and H). We used murine AML models to evaluate the therapeutic efficacy of LNT cell/DOX. In tumor-bearing C57BL/6J mice, tumor growth was monitored by bioluminescence signals upon treatment (fig. S7, A to C). In this leukemia model, although LNT cells alone exhibited no antitumor effects, LNT cell/DOX treatment reduced the tumor growth compared with control treatments (fig. S7, D to H).

Tumor cell lysates can function as cancer vaccines and initiate tumor-specific immune responses (27, 28). We hypothesized that LNT cells can enhance the antigen uptake and maturation of antigen-presenting cells (APCs). LNT cells cocultured with dendritic cells (DCs) caused their maturation as assessed by up-regulation of CD40, CD80, CD86, and major histocompatibility complex II (MHC-II) (fig. S8A). Moreover, CD4+ T cells and CD8+ T cells increased in the peripheral blood of the mice receiving LNT cells and the adjuvant of monophosphoryl lipid A (MPLA) (fig. S8B). DC maturation and T cell activationrelated cytokines, including interferon- (IFN-), tumor necrosis factor (TNF-), and interleukin-6 (IL-6), were also detected in mice treated with LNT cell and adjuvant (fig. S8C). We next evaluated the antitumor efficacy of LNT cell/DOX with adjuvant in leukemia-bearing mice. As demonstrated in Fig. 3 (A and B), bioluminescence of AML cancer cells increased rapidly in untreated mice, while AML had been partially inhibited after DOX or LNT cell and adjuvant treatment. AML cells were almost completely eliminated in mice treated with LNT cell/DOX and adjuvant up to 21 days after tumor inoculation (Fig. 3B). Quantitative analysis of tumor bioluminescence and survival analysis also demonstrated superior therapeutic activity of LNT cell/DOX combined with adjuvant (Fig. 3, C to E). Increased serum levels of IFN- and TNF- (Fig. 3, F and G), as well as elevation of CD3+ T cell and CD8+ T cells, supported the occurrence of boosted immunity in the mice receiving LNT Cell/DOX and adjuvant treatment (Fig. 3, H and I).

(A) Schematic of the treatment model. (B) AML progression in vivo as indicated by bioluminescence signal expressed by luciferase tagged C1498 cells during different treatments (G1, saline; G2, DOX; G3, LNT cell + adjuvant; G4, LNT cell/DOX + adjuvant). (C) Quantified bioluminescence of different treatment groups. (D) Bioluminescence intensity of treated mice on day 21 (n = 6). (E) Survival of the mice of different treatment groups (n = 6). Serum cytokine levels of IFN- (F), TNF- (G), and proportion of peripheral CD3+ T cells (H) and CD8+ T cells (I) on day 13 (n = 6). Data are presented as means SD. (D and F to I). Statistical significance was calculated via ordinary one-way ANOVA (D and F to I) and log-rank (Mantel-Cox) test (E). *P < 0.05, **P < 0.01, ***P < 0.001.

We further evaluated the efficacy of LNT cells as a prophylactic cancer vaccine. Mice were first immunized at 21, 14, and 7 days before challenge with live C1498 cells. The onset of AML in mice was prevented in mice preimmunized with LNT cells and adjuvant (Fig. 4, A to C). Quantitative data also revealed that the tumor bioluminescence intensity of the group of LNT cells with adjuvant was substantially lower than control groups (Fig. 4D). Moreover, 71% of the mice treated with LNT cells and adjuvant were tumor free 90 days after tumor challenge, while all control mice died by day 34 (Fig. 4E). Serum levels of IFN-, TNF-, IL-12, and IL-6 were significantly increased in mice treated with LNT cells and adjuvant (Fig. 4F), indicating that a prompt immune response was triggered upon tumor cell inoculation. In addition, CD3+ T cells and CD8+ T cells were significantly increased in the peripheral blood of mice vaccinated with LNT cells and adjuvant (Fig. 4, G and H, and fig. S9).

(A) Schematic of the treatment model. Bioluminescence images (B) and quantified bioluminescence (C) of the mice preimmunized with different treatment formulations (G1, saline; G2, adjuvant; G3, LNT cell + adjuvant). (D) Bioluminescence intensity of treated mice on day 47 (n = 5 for G1 and G2 for one mice died before day 47; n = 7 for G3). (E) Survival of the mice after tumor challenge (n = 6 for G1 and G2; n = 7 for G3). (F) Serum cytokine levels 3 days after challenge of live C1498 cells (n = 6 for G1 and G2; n = 7 for G3). (G) Representative flow cytometry images of CD3+ T cells (left) and proportion of peripheral CD3+ T cells (right) on day 24 (n = 6 for G1 and G2; n = 7 for G3). (H) Representative flow cytometry images of CD8+ T cells (left) and corresponding proportion of peripheral CD8+ T cell gating on CD3+ T cells (right) on day 24 (n = 6 for G1 and G2; n = 7 for G3). Data are presented as means SD. (D and F to H). Statistical significance was calculated via ordinary one-way ANOVA (D and F to H) and log-rank (Mantel-Cox) test (E), *P < 0.05, **P < 0.01, ***P < 0.001.

In this study, we demonstrated the feasibility, efficacy, and safety of tumor dead cells used as a drug-targeting carrier and tumor vaccine for cancer therapy. Compared with the synthetic materialmediated delivery vehicles, cell-based carriers show unique targeting capacities and can bypass biological barriers (15, 29). AML cells originate in the bone marrow and naturally exhibit similar bone marrow homing capabilities as HSCs (22, 30, 31), rendering them suitable to be used as cellular drug carriers for AML therapy. However, it remains essential to develop strategies allowing the elimination of AML tumorigenicity while transiently preserving cellular integrity to deliver the payload at the tumor site. We therefore proposed to use the dead but functional AML cells as the drug carrier.

Usually, the structure of the live cells can disintegrate upon dying with the loss of proteins and cytokines (32). In addition, external stimuli that could induce cell death, such as heat or radiation, will deactivate proteins as well (33, 34). Our data support the concept that cryo-shocked tumor cells obtained by rapid immersion of live cells in liquid nitrogen lose tumorigenicity while preserving transiently the integrity of the cell structure, which is critical for the drug loading and cargo release. Furthermore, certain critical functional proteins that include CD44 and CXCR4 were retained in LNT cells. CD44 can interact with hyaluronic acid that is highly expressed in the endosteum of bone marrow (18). CXCR4 enables cells to migrate toward the chemokine stromal cellderived factor 1 (SDF-1) that is constitutively produced by the osteoblasts and stromal cells (23). CD44 and CXCR4 are two important adhesion receptors mediating AML cells homing toward bone marrow (35). Retention of both CD44 and CXCR4 in LNT cells, even if at reduced levels compared with live cells, is likely critical to promote their bone marrow homing. The proposed LNT-based strategy is simple and straightforward from a manufacturing point of view. Tumor cells in the case of liquid tumors can be readily collected in large quantity, for example, by leukapheresis. Similarly, for solid tumors, multiple devises are currently available to generate single cell suspension from resected tumors or tumor biopsies. The process of cell shocking in liquid nitrogen is also feasible to standardize in good manufacturing practice conditions.

We evaluated the proliferation and tumorigenicity of LNT tumor cells both in vitro and in vivo. The data that all mice treated with LNT cells exhibited no obvious side effects and no leukemia growth was recorded for 6 months after inoculation of LNT C1498 cells support at least in our mouse model the safety of the proposed strategy. After exposure to liquid nitrogen, the cellular membrane of LNT cells becomes permeable. While live cells require treatment with cell membrane detergent to obtain intracellular staining, LNT cells do not require this treatment, indicating the loss of long-term integrity of the cell membrane, which is essential to cell survival. However, our experiments demonstrate that liquid nitrogen treatment does not impair the capacity of LNT cells to function as drug carrier and tumor vaccine. The cryo-shocking technique could be a platform technology in cell bioengineering and could be applicable to various cell types. Here, we have further tested feasibility in 4T1 tumor cells (fig. S10). Regarding the potential impact in clinical use, the safety of LNT tumor cells, besides C1498 cells adopted in this work, should be evaluated thoroughly in other experimental animal models. In addition, the application of LNT cells to serve as drug carriers of other therapeutics, such as immune checkpoint inhibitors (36), is worth investigating.

In summary, we engineered LNT tumor cells to serve simultaneously as a drug delivery carrier and cancer vaccine. The simple liquid nitrogen treating process abrogates the tumorigenicity of tumor cells but preserves the integrity of their cellular structure. This in turn allows the possibility to load LNT cells with chemotherapy drugs and preserves the homing capacity of these cells to the tumor site. LNT cells in combination with adjuvant could elicit both therapeutic and protective immune antitumor responses and may avoid the complex quality control associated with isolated cells and synthesized materialbased vehicles and enable large-scale production for clinical use.

The aim of this study was to use the cryo-shocked tumor cells as a kind of drug-targeting carrier and tumor vaccine for chemo-immunotherapy in the treatment of AML. After treating the live cells in liquid nitrogen, the cellular structure of the cryo-shocked cells was observed. The proliferation behavior, in vivo tumorigenicity, and targeting capability toward the bone marrow of the cryo-shocked cells were assessed. In vivo antitumor efficacy was analyzed in an AML model by intravenously injecting C1498 cells in C57BL/6J mice. Mice were randomly assigned to groups based on body weights. After different treatments, the mice were captured by in vivo imaging system (IVIS) to evaluate in vivo tumor progression. Survival curves, immune cell proportions, and cytokine levels were determined according to previous experimental experience. Specific information about treatment groups, sample numbers, and data analysis was denoted in the figure captions.

Doxorubicin hydrochloride was purchased from Fisher Scientific Co. (D4193; purity, >95%). Noncontrolled-rate cell cryopreservation medium was bought from Cyagen Co. (NCRC-10001-50). AML cell line C1498 was purchased from the American Type Culture Collection (ATCC). Luciferase and DsRed tagged C1498 cell line was provided by B. Blazar of the University of Minnesota. The cells were cultured in 90% Dulbeccos modified Eagles medium (Gibco) and 10% fetal bovine serum (Gibco) with penicillin (200 U ml1) and streptomycin (200 U ml1) (Gibco). The cells were passaged every 1 to 2 days. C57BL/6J mice (4 to 6 weeks, female) were purchased from the Jackson laboratory. All animal tests complied with the animal protocol approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles.

C1498 cells were centrifuged at 250g for 3 min and suspended in noncontrolled-rate cell cryopreservation medium at a cell density of 1 106 to 1 107 ml1. The cell-containing medium was immersed in liquid nitrogen for 12 hours. Before use, the medium was thawed at 37C and LNT cells were pelleted at 500g for 3 min. After washing with PBS solution (pH 7.4), LNT cells were suspended in PBS and kept at 4C. For preparation of DOX-loaded LNT cells, the LNT cells were suspended in DOX containing PBS. After incubation for 2 hours, the medium was centrifuged at 500g for 5 min and the pellets were DOX-loaded LNT cells.

The AML model was established by intravenous injection of 5 106 C1498 cells on day 0. On day 8 and day 15, saline, LNT cell + adjuvant, free DOX, and LNT cell/DOX + adjuvant were administrated intravenously with DOX dose of 5 mg/kg and adjuvant (MPLA) 20 g per mouse. Specifically, MPLA was intravenously injected 10 hours after injection of LNT cell or LNT cell/DOX. The bioluminescence images of mice were captured every 3 days. The exposure time was 2 min. On day 13, 400 l of blood was collected via the orbital vein. Blood (200 l) was treated with ammonium-chloride-potassium (ACK) buffer and centrifuged at 800g for 8 min to obtain pellets of white blood cells. After staining with BV421-CD3, PE-CD4, and APC-CD8, the samples were analyzed by flow cytometry. Another 200 l of blood in blood serum collection tubes (BD Microtainer 365967) was centrifuged at 3000 rpm for 10 min. The upper serum was detected with the following enzyme-linked immunosorbent assay kits: IFN- (BioLegend 430804) and TNF- (BioLegend 430904).

The results were presented as means SD or mean standard error of the mean (means SEM) as indicated. The data were compared by Students t test between two groups and ordinary one-way analysis of variance (ANOVA) for three or more groups. The survival curves were analyzed via the log-rank (Mantel-Cox) test. All statistical analyses were conducted by the GraphPad Prism software. The threshold of a statistically significant difference was defined as P < 0.05.

Acknowledgments: We acknowledge B. Blazar at the University of Minnesota for providing the luciferase and DsRed tagged C1498 cell line. Funding: This work was supported by the NIH (R01 CA234343-01A1) and grants from the start-up packages of UCLA. Author contributions: Z.G. and T.C. proposed the conception of the project. T.C. and H.L. performed all the experiments and collected the data with the help of G.C., Z.W., J.W., P.A., and Y.T. All authors analyzed the data and contributed to the writing of the manuscript, discussed the results and implications, and edited the manuscript at all stages. Competing interests: Z.G. and T.C. are inventors on a U.S. patent application related to this work filed by University of California, Los Angeles (no. 63/094,034, filed [Oct 20th, 2020]). Z.G. is a scientific cofounder of ZenCapsule Inc. The authors declare that they have no other competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors.

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