Category Archives: Genetic Therapy

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

Dec. 15, 2020 11:00 UTC

MENLO PARK, Calif.--(BUSINESS WIRE)-- Avellino announced today the appointment of three new members to its Board of Directors. In addition, the company announced a new Chief Executive Officer and President, and Chief Financial Officer and Treasurer. All appointments are effective December 2020.

The Board of Directors has been further strengthened by the appointment of Aimee S. Weisner, Esq.; Richard Gannotta, NP, DHA, FACHE; and William Stasior, MS, PhD. Joining the executive team is current Board member Jim Mazzo as CEO and President, and Cyril Allouche as CFO and Treasurer.

Avellino Group Chairman Gene Lee said, All of our newest Board members embody the spirit and ingenuity of Avellino and bring such broad talent, expertise, and energy to the table. We are very fortunate to have them by our side to help us continue to grow as a company. And with Jim moving into a senior executive leadership position, his immeasurable business experience with scientific acumen will enhance our ability to continue to lead the way in delivering best-in-class personalized genetic and molecular diagnostics, data, and therapeutics. Also, we are excited to welcome Cyril to our leadership team. His depth of business experience across multiple industries and preparing companies for entering public markets will be a perfect complement to the scientific expertise we have fostered at Avellino.

Board of Directors Appointments

Aimee S. Weisner, Esq. is an experienced independent director in the medical device, pharmaceutical and biotech spaces, and she brings significant expertise as a corporate medtech executive and attorney. Most recently, from 2011 to 2019, Ms. Weisner served as Corporate Vice President, General Counsel of Edwards Lifesciences Corporation.

Richard Gannotta, NP, DHA, FACHE is a recognized leader in the health sector with service in CEO / President and executive roles in some of the nations most prominent academic and public health systems and a leading global medical technology company. In addition, Dr. Gannotta is Senior Lecturer at the NYU Wagner Graduate School of Public Service where his area of focus is on the management of healthcare organizations and health policy.

William Stasior, MS, PhD has established himself as a creative innovator with technical expertise at Silicon Valleys most recognizable technology companies. Dr. Stasior currently serves as Corporate Vice President, Technology, and a member of the Office of the Chief Technology Officer at Microsoft. Prior to joining Microsoft, he was for many years the Vice President, Artificial Intelligence, at Apple and head of Apples Siri division. Among many of his career accomplishments, Dr. Stasior also served as the Vice President of Amazon Search and was CEO of Amazon Silicon Valley subsidiary A9.com. Prior to joining the Board, Mr. Stasior provided guidance to Avellino as part of the Executive Advisory Committee.

Chief Executive Officer and President

Avellino appoints Jim Mazzo as the new CEO and President. Mr. Mazzo is one of the ophthalmic industrys best known and most respected business leaders with over 38 years of proven experience. His global reputation for building and running world-class organizations is based on 22 years leading Allergans North American and European eye care organizations. His many accomplishments and contributions to the healthcare, business and educational communities include serving as Board Chairman for AdvaMed as well as Vice-Chairman and Trustee for Chapman University and the University of San Diego.

Chief Financial Officer and Treasurer

Avellino also welcomes Cyril Allouche to the executive team as its new Chief Financial Officer and Treasurer. Mr. Allouche brings to Avellino over 20 years of experience in finance leadership in both public and pre-IPO companies, including diagnostics and biopharmaceutical. He most recently served as CFO at Dermavant Sciences and held finance leadership roles at Revance Therapeutics and CareDx. He also spent over a decade at PricewaterhouseCoopers in Audit and Transaction services.

The inclusion of Aimee, Richard, and William provides expanded leadership and broader operational, digital, marketing, and commercialization expertise that will surely complement our executive team. Along with Cyrils deep and extensive experience in leading the financial operations of healthcare businesses, Avellino will continue to grow our genetic and molecular diagnostic tests pipeline and flourish as a company, said Avellino Group Chairman Gene Lee.

Added newly appointed CEO Jim Mazzo: Its exciting times here at Avellino, all of the additions to the boardroom along with the changes taking place at the senior executive level shows that we are set up for success with unlimited potential for tremendous growth. Considering this, and the positive impact Avellino has had in providing testing during the pandemic and their efforts to fight blindness since their inception, and the advances they will bring to healthcare in the future, joining the senior management team was an easy decision for me to make.

About Avellino

Avellino Lab USA, Inc. is a global leader in gene therapy and molecular diagnostics and is at the forefront of precision medicine for eye care. The company is a proud member of the California State COVID-19 Testing Taskforce, which is focused on the expansion of CoV2 testing and the reduction of testing turn-around times (TAT). Beyond the AvellinoCoV2 test, Avellino recently launched AvaGen, the worlds first DNA test to confirm the presence of genetic indicators that are positively associated with corneal dystrophies and keratoconus genetic risk factors. The company will also soon launch an infectious disease panel of diagnostic tests. Beyond diagnostics, the company is also pioneering CRISPR gene editing to manage and potentially cure inherited diseases. Avellino is headquartered in Silicon Valley, California, with operations in Korea, Japan, China, and the UK.

To learn more about Avellino, please visit http://www.avellino.com.

View source version on businesswire.com: https://www.businesswire.com/news/home/20201215005070/en/

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Avellino Expands Its Board of Directors and Welcomes New Executive Management Members - BioSpace

An innovative research study has been offered by Futuristic Reports, offering a comprehensive analysis of the Global Gene Therapy Market where users can get an advantage from the comprehensive market research report with all the essential useful information. This is the newest report, covering the existing COVID-19 impact on the Gene Therapy market. It has fetched along with numerous changes in market conditions. This segment also provides the Gene Therapy scope of different applications and types that can potentially influence the future market. The comprehensive statistics are based on current trends and historical milestones.

This report also delivers an analysis of production volume about the global Gene Therapy market and each type from 2020 to 2026. The Gene Therapy report explicitly features the market share, company profiles, regional viewpoint, product portfolio, recent developments, newest strategic analysis, key players in the market, deals, circulation chain, manufacturing, production, and newest market entrants. The existing Gene Therapy market players, brand value, popular products, demand and supply, and other significant factors identified with the market help players will better understand the market scenario.

Impact of COVID-19 on Gene Therapy Market

The report also contains the effect of the ongoing worldwide pandemic, i.e., COVID-19, on the Gene Therapy Market and what the future holds for it. It offers an analysis of the impacts of the epidemic on the international market. The epidemic has immediately interrupted the requirement and supply series. The report also assesses the economic effect on firms and economic demands. Futuristic Reports has accumulated advice from several delegates of this Gene Therapy business and has engaged from the secondary and primary research to extend the customers with strategies and data to combat industry struggles throughout and after the COVID-19 pandemic.

Some of the key players operating in this market include:

(Kite Pharma Inc., Spark Therapeutics Inc., Novartis, GlaxoSmithKline PLC, Applied Genetic Technologies Corporation, NewLink Genetics Corp, Transgene SA, Oxford BioMedica, Genethon, Bluebird biInc.)

Based on Product Type, Gene Therapy market report displays the production, profits, cost, and market segment and growth rate of each type, covers:

By Gene Type Deficiency Tumor Suppressor Antigen Cytokine Receptors Suicide Growth factors Others

Based on end users/applications, the Gene Therapy market report focuses on the status and viewpoint for major applications/end users, sales volume, market share, and growth rate for each application. This can be divided into:

Rare Diseases Neurological Disorders Oncological Disorders Cardiovascular Diseases Infectious disease Other

The report offers a comprehensive assessment of the progression and other Gene Therapy market features in significant regions, including South Korea, Taiwan, North America, Europe, Canada, Germany, France, Southeast Asia, Mexico, and Brazil, Pacific, and Latin America. U.S., U.K., Italy, Russia, China, Japan, etc.

Features the following key factors:

Some of the Key Questions Answered in this Report:

Futuristic Reports

Name: Mark RiveraTel: +1-408-520-9037Email: [emailprotected]

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Gene Therapy Market Growth, Developments and Forecast by 2026 - LionLowdown

CAMBRIDGE, Mass.--(BUSINESS WIRE)--bluebird bio, Inc. (Nasdaq: BLUE) announced that new data from Group C of its ongoing Phase 1/2 HGB-206 study of investigational LentiGlobin gene therapy (bb1111) for adult and adolescent patients with sickle cell disease (SCD) show a complete elimination of severe VOEs and VOEs between six and 24 months of follow-up. These data are being presented at the 62nd American Society of Hematology (ASH) Annual Meeting and Exposition, taking place virtually from December 5-8, 2020.

Now with more than two years of data, we continue to observe promising results in our studies of LentiGlobin for SCD that further illustrate its potential to eliminate the symptoms and devastating complications of sickle cell disease. Consistently achieving the complete resolution of severe vaso-occlusive events (VOEs) and VOEs between Month 6 and Month 24 follow-up is unprecedented other than with allogeneic stem cell transplantation. Importantly, our data show the potential for LentiGlobin for SCD to produce fundamentally disease-modifying effects with sustained pancellular distribution of gene therapy-derived anti-sickling HbAT87Q and improvement of key markers of hemolysis that approach normal levels, said David Davidson, M.D., chief medical officer, bluebird bio. In addition to these clinical outcomes, for the first time with a gene therapy we now have patient-reported outcomes through the validated PROMIS-57 tool, showing reduction in pain intensity at 12 months after treatment with LentiGlobin for SCD. These results provide insight into the potential real-life impact LentiGlobin for SCD may offer patients.

SCD is a serious, progressive and debilitating genetic disease. In the U.S., the median age of death for someone with sickle cell disease is 43 46 years. SCD is caused by a mutation in the -globin gene that leads to the production of abnormal sickle hemoglobin (HbS). HbS causes red blood cells to become sickled and fragile, resulting in chronic hemolytic anemia, vasculopathy and unpredictable, painful VOEs.

In the HGB-206 study of LentiGlobin for SCD, VOEs are defined as episodes of acute pain with no medically determined cause other than a vaso-occlusion, lasting more than two hours and severe enough to require care at a medical facility. This includes acute episodes of pain, acute chest syndrome (ACS), acute hepatic sequestration and acute splenic sequestration. A severe VOE requires a 24-hour hospital stay or emergency room visit or at least two visits to a hospital or emergency room over a 72-hour period, with both visits requiring intravenous treatment.

LentiGlobin for SCD was designed to add functional copies of a modified form of the -globin gene (A-T87Q-globin gene) into a patients own hematopoietic (blood) stem cells (HSCs). Once patients have the A-T87Q-globin gene, their red blood cells can produce anti-sickling hemoglobin (HbAT87Q) that decreases the proportion of HbS, with the goal of reducing sickled red blood cells, hemolysis and other complications.

As a hematologist, I regularly see the debilitating effects of pain events caused by sickle cell disease. Pain has an overwhelmingly negative impact on many facets of my patients lives and can lead to prolonged hospitalizations, said presenting study author Alexis A. Thompson, M.D., professor of pediatrics at Northwestern University Feinberg School of Medicine and head of hematology at Ann and Robert H. Lurie Childrens Hospital of Chicago. The results observed with LentiGlobin gene therapy for SCD include the complete elimination of severe vaso-occlusive pain episodes, which is certainly clinically meaningful, but also for the first time, we have documented patients reporting that they are experiencing improved quality of life. This degree of early clinical benefit is extraordinarily rewarding to observe as a provider."

As of the data cut-off date of August 20, 2020, a total of 44 patients have been treated with LentiGlobin for SCD in the HGB-205 (n=3) and HGB-206 (n=41) clinical studies. The HGB-206 total includes: Groups A (n=7), B (n=2) and C (n=32).

HGB-206: Group C Updated Efficacy Results

The 32 patients treated with LentiGlobin for SCD gene therapy in Group C of HGB-206 had up to 30.9 months of follow-up (median of 13.0; min-max: 1.1 30.9 months).

In patients with six or more months of follow-up whose hemoglobin fractions were available (n=22), median levels of gene therapy-derived anti-sickling hemoglobin, HbAT87Q, were maintained with HbAT87Q contributing at least 40% of total hemoglobin at Month 6. At last visit reported, total hemoglobin ranged from 9.6 15.1 g/dL and HbAT87Q levels ranged from 2.7 8.9 g/dL. At Month 6, the production of HbAT87Q was associated with a reduction in the proportion of HbS in total hemoglobin; median HbS was 50% and remained less than 60% at all follow-up timepoints. All patients in Group C were able to stop regular blood transfusions by three months post-treatment and remain off transfusions as of the data cut-off.

Nineteen patients treated in Group C had a history of severe VOEs, defined as at least four severe VOEs in the 24 months prior to informed consent (annualized rate of severe VOE min-max: 2.0 10.5 events) and at least six months follow-up after treatment with LentiGlobin for SCD. There have been no reports of severe VOEs in these Group C patients following treatment with LentiGlobin for SCD. In addition, all 19 patients had a complete resolution of VOEs after Month 6.

Hemolysis Markers

In SCD, red blood cells become sickled and fragile, rupturing more easily than healthy red blood cells. The breakdown of red blood cells, called hemolysis, occurs normally in the body. However, in sickle cell disease, hemolysis happens too quickly due to the fragility of the red blood cells, which results in hemolytic anemia.

Patients treated with LentiGlobin for SCD in Group C demonstrated near-normal levels in key markers of hemolysis, which are indicators of the health of red blood cells. Lab results assessing these indicators were available for the majority of the 25 patients with 6 months of follow-up.

The medians for reticulocyte counts (n=23), lactate dehydrogenase (LDH) levels (n=21) and total bilirubin (n=24) continued to improve compared to screening values and stabilized by Month 6. In patients with Month 24 data (n=7), these values approached the upper limit of normal by Month 24. These results continue to suggest that treatment with LentiGlobin for SCD may improve biological markers to near-normal levels for SCD.

Pancellularity

As previously reported, assays were developed by bluebird bio to enable the detection of HbAT87Q and HbS protein in individual red blood cells, as well as to assess if HbAT87Q was pancellular, or present throughout all of a patients red blood cells. In 25 patients with at least six months of follow-up, on average, more than 80% of red blood cells contained HbAT87Q, suggesting near-complete pancellularity of HbAT87Q distribution and with pancellularity further increasing over time.

HGB-206: Improvements in Health-Related Quality of Life

Health-related quality of life (HRQoL) findings in Group C patients treated with LentiGlobin for SCD in the HGB-206 study were generated using the Patient Reported Outcomes Measurement Information System 57 (PROMIS-57), a validated instrument in SCD.

Data assessing pain intensity experienced by nine Group C patients were analyzed according to baseline pain intensity scores relative to the general population normative value: 2.6 on a scale of 0-10, where 10 equals the most intense pain. Data were assessed at baseline, Month 6 and Month 12.

Of the five patients with baseline scores worse than the population normative value average, four demonstrated clinically meaningful reductions in pain intensity at Month 12; the group had a mean score of 6.0 at baseline and a mean score of 2.4 at Month 12. Of the four patients with better than or near population normative values at baseline, two reported improvement and two remained stable with a mean score of 2.3 at baseline and 0.8 at Month 12.

HGB-206: Group C Safety Results

As of August 20, 2020, the safety data from Group C patients in HGB-206 remain generally consistent with the known side effects of hematopoietic stem cell collection and myeloablative single-agent busulfan conditioning, as well as underlying SCD. One non-serious, Grade 2 adverse event (AE) of febrile neutropenia was considered related to LentiGlobin for SCD. There were no serious AEs related to LentiGlobin for SCD.

One patient with significant baseline SCD-related and cardiopulmonary disease died 20 months post-treatment; the treating physician and an independent monitoring committee agreed his death was unlikely related to LentiGlobin for SCD and that SCD-related cardiac and pulmonary disease contributed.

LentiGlobin for SCD Data at ASH

The presentation of HGB-206 Group C results and patient reported outcomes research are now available on demand on the ASH conference website:

About HGB-206

HGB-206 is an ongoing, Phase 1/2 open-label study designed to evaluate the efficacy and safety of LentiGlobin gene therapy for sickle cell disease (SCD) that includes three treatment cohorts: Groups A (n=7), B (n=2) and C (n=32). A refined manufacturing process designed to increase vector copy number (VCN) and further protocol refinements made to improve engraftment potential of gene-modified stem cells were used for Group C. Group C patients also received LentiGlobin for SCD made from HSCs collected from peripheral blood after mobilization with plerixafor, rather than via bone marrow harvest, which was used in Groups A and B of HGB-206.

About LentiGlobin for SCD (bb1111)

LentiGlobin gene therapy for sickle cell disease (bb1111) is an investigational treatment being studied as a potential treatment for SCD. bluebird bios clinical development program for LentiGlobin for SCD includes the completed Phase 1/2 HGB-205 study, the ongoing Phase 1/2 HGB-206 study, and the ongoing Phase 3 HGB-210 study.

The U.S. Food and Drug Administration granted orphan drug designation, fast track designation, regenerative medicine advanced therapy (RMAT) designation and rare pediatric disease designation for LentiGlobin for SCD.

LentiGlobin for SCD received orphan medicinal product designation from the European Commission for the treatment of SCD, and Priority Medicines (PRIME) eligibility by the European Medicines Agency (EMA) in September 2020.

bluebird bio is conducting a long-term safety and efficacy follow-up study (LTF-307) for people who have participated in bluebird bio-sponsored clinical studies of LentiGlobin for SCD. For more information visit: https://www.bluebirdbio.com/our-science/clinical-trials or clinicaltrials.gov and use identifier NCT04628585 for LTF-307.

LentiGlobin for SCD is investigational and has not been approved in any geography.

About bluebird bio, Inc.

bluebird bio is pioneering gene therapy with purpose. From our Cambridge, Mass., headquarters, were developing gene and cell therapies for severe genetic diseases and cancer, with the goal that people facing potentially fatal conditions with limited treatment options can live their lives fully. Beyond our labs, were working to positively disrupt the healthcare system to create access, transparency and education so that gene therapy can become available to all those who can benefit.

bluebird bio is a human company powered by human stories. Were putting our care and expertise to work across a spectrum of disorders: cerebral adrenoleukodystrophy, sickle cell disease, -thalassemia and multiple myeloma, using gene and cell therapy technologies including gene addition, and (megaTAL-enabled) gene editing.

bluebird bio has additional nests in Seattle, Wash.; Durham, N.C.; and Zug, Switzerland. For more information, visit bluebirdbio.com.

Follow bluebird bio on social media: @bluebirdbio, LinkedIn, Instagram and YouTube.

LentiGlobin and bluebird bio are trademarks of bluebird bio, Inc.

Forward-Looking Statements

This release contains forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995. Any forward-looking statements are based on managements current expectations of future events and are subject to a number of risks and uncertainties that could cause actual results to differ materially and adversely from those set forth in or implied by such forward-looking statements. These risks and uncertainties include, but are not limited to: regarding the potential for LentiGlobin for Sickle Cell Disease to treat SCD; the risk that the efficacy and safety results from our prior and ongoing clinical trials will not continue or be repeated in our ongoing or planned clinical trials; the risk that the current or planned clinical trials of our product candidates will be insufficient to support regulatory submissions or marketing approval in the United States and European Union; the risk that regulatory authorities will require additional information regarding our product candidates, resulting in delay to our anticipated timelines for regulatory submissions, including our applications for marketing approval; and the risk that any one or more of our product candidates, will not be successfully developed, approved or commercialized. For a discussion of other risks and uncertainties, and other important factors, any of which could cause our actual results to differ from those contained in the forward-looking statements, see the section entitled Risk Factors in our most recent Form 10-Q, as well as discussions of potential risks, uncertainties, and other important factors in our subsequent filings with the Securities and Exchange Commission. All information in this press release is as of the date of the release, and bluebird bio undertakes no duty to update this information unless required by law.

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Treatment with Investigational LentiGlobin Gene Therapy for Sickle Cell Disease (bb1111) Results in Complete Elimination of SCD-Related Severe...

This weekend brought some really significant news in the long-running effort to use gene editing to treat human disease. As most readers will have heard, Boston Children's Hospital and a Vertex/CRISPR effort both published papers in the NEJM addressing sickle-cell anemia and beta-thalassemia.

These diseases have long been linked when it comes to gene therapy ideas, because both of them have defects in the hemoglobin protein as their cause. And it's long been thought that both could be treated by getting adults to re-express the fetal hemoglobin protein - it's on a different gene entirely, and thus does not have any of the genetic problems that affect the adult hemoglobin gene. The normal course of events is for babies to stop expressing the fetal form and switch over to "regular" hemoglobin, and it's been worked out that a particular transcription factor called BCL11a is a key player in that transcriptional repression of the fetal hemoglobin gene. That plays right into the usual way that we tend to think about therapeutic possibilities: whether it's enzymes, receptors, or expression of whole proteins, we have a lot more tools to mess things up and interrupt processes than we have to make them run faster or better. So the possibility of interrupting BCL11a's function has been a tempting one for many years.

It's hard to do by traditional means, though. (Full disclosure: I have, at different times in my career, been involved with such efforts, but none have ever come near the clinic.) Transcription factors are notoriously hard to get a handle on with small molecule therapeutics, and many unsuccessful runs have been taken at BCL11a ligands to try to interrupt its functions in one way or another. My general impression is that the protein doesn't much care about recognizing small-molecule ligands (and it's far from the only one in that category, for sure). You'd think that if you ran a few hundred thousand (or a few million) various molecules past any given protein that you'd find a few of them that bind to it, but that assumption is too optimistic for most transcription factors. You're also going to have a hard row to hoe (to use an old Arkansas expression) if you try to break up their interactions with their DNA binding sites: a significant amount of capital has gone down the chute trying to get that to work, with (as far as I can tell) not much to show for it.

There's another complication: BCL11a has a lot of other functions. Every protein has a lot of other functions, but for transcription factors, the issue can be especially fraught. If you had a small molecule that really did interfere with its activity, what would happen if you just took a stiff dose of it? Probably a number of things, including some interesting (and not necessarily welcome) surprises. There have been a number of ideas about how to get around this problem, but a problem it is.

So it's on to biological mechanisms. The BCH team reports on using RNA interference to do the job - they get cells to express a short hairpin RNA that shuts down production of BCL11a protein, with some microRNA work to target this to the right cell lines. And the Vertex (NASDAQ:VRTX)/CRISPR (NASDAQ:CRSP) team, naturally, uses CRISPR itself to go in and inactivate the BCL11a gene directly. Both approaches take (and have to take) a similar pathway, which is difficult and expensive, but still the best shot at such therapies that we have. You want the fetal hemoglobin expressed in red blood cells, naturally, and red blood cells come from CD34+ stem cells in the bone marrow. Even if you haven't thought about this, you might see where it's going: you take a bone marrow sample, isolate these cells, and then do your genetic manipulation to them ex vivo.

Once you've got a population of appropriately re-engineered cells ready to go, you go kill off the bone marrow in the patient and put the reworked cells back in, so they're the only source there for red blood cells at all. A bone marrow transplant, in other words - a pretty grueling process, but definitely not as much as having some sort of blood-cell-driven cancer (where the therapy uses compatible donor cells from someone else without such a problem), or as much as having full-on sickle cell disease or tranfusion-dependent thalassemia.

You can also see how this is a perfect setup for gene therapy: there's a defined population of cells that you need to treat, which are available in a specific tissue via a well-worked-out procedure. The problem you're trying to correct is extremely well understood - in fact, it was the first disease ever characterized (by Linus Pauling in 1949) as purely due to a genetic defect . And the patient's own tissue is vulnerable to chemotherapy agents that will wipe out the existing cell population, in another well-worked-out protocol, giving the newly reworked cells an open landscape to expand in. You have the chance for a clean swap on a defined target, which is quite rare. In too many other cases the problem turns out to involve a fuzzy mass of genetic factors and environmental ones, none of which by themselves account for the disease symptoms, or the tissue doesn't allow you to isolate the defective cells easily or doesn't allow you to clear them out for any new ones you might generate, and so on.

Both the Vertex/CRISPR and BCH techniques seem to work - and in fact, to work very well. There are now people walking around, many months after these treatments, who were severely ill but now appear to be cured. That's not a word we get to use very often. They are producing enough fetal hemoglobin, more than enough to make their symptoms completely disappear - no attacks, no transfusions, just normal life. And so far there have been no side effects due to the altered stem cells. An earlier strategy from Bluebird (involving addition of a gene for a modified adult hemoglobin) also seems to be holding up.

These are revolutionary proofs of concept, but at the same time, they are not going to change the course of these diseases in the world - not right now, anyway. Bone marrow transfusion is of course a complex process that costs a great deal and can only be done in places with advanced medical facilities. But what we've established is that anything that can cause fetal hemoglobin to be expressed should indeed cure these diseases - that idea has been de-risked. As has the general idea of doing such genetic alteration in defined adult tissues (either RNA interference or CRISPR).

From here, we try to make these things easier, cheaper and more general, to come up with new ways of realizing these same goals now that we know that they do what we hoped that they would. This work is already underway - new ways to target the affected cell populations rather than flat-out chemotherapy assault, new ways to deliver the genetically altered cells (or to produce them "on site" in the patients), ways to make the switchover between the two more gradual, and so on. There are lot of possible ways, and we now know where we're going.

Disclosure: None

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Editor's Note: The summary bullets for this article were chosen by Seeking Alpha editors.

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Gene Therapy, Absolutely And For Real - Seeking Alpha

NEW YORK & BRISBANE, Calif.--(BUSINESS WIRE)--Pfizer Inc. (NYSE: PFE) and Sangamo Therapeutics, Inc. (Nasdaq: SGMO), a genomic medicines company, today announced updated follow-up data from the Phase 1/2 Alta study of giroctocogene fitelparvovec (SB-525 or PF-07055480), an investigational gene therapy for patients with severe hemophilia A. These data are being presented today at the 62nd American Society for Hematology Annual meeting taking place virtually from December 5th - 8th. The oral presentation slides, which include follow-up data up to 85 weeks for the longest treated patient, are available on Sangamos website in the Investors and Media section under Events and Presentations.

All five patients in the high dose 3 x 1013 vg/kg cohort have had at least one year of follow-up and showed sustained factor VIII (FVIII) activity levels, with a group median FVIII activity of 56.9% and a group geometric mean FVIII activity of 70.4% via chromogenic assay from week 9 to 52. Steady-state FVIII activity was achieved for all patients in the 3 x 1013 vg/kg cohort within 9 weeks of treatment with giroctocogene fitelparvovec, with no bleeding events and no FVIII infusions (beyond 3 weeks post-infusion) within the first year. As of the cutoff date of August 31, 2020, one patient had one target joint bleed requiring FVIII therapy, occurring after week 52.

It is promising to see how quickly all five patients in the 3 x 1013 vg/kg cohort achieved steady-state FVIII activity levels, with no bleeding events and no factor usage within the first year and only one target joint bleed after 52 weeks, said Andrew D. Leavitt, MD, Professor of Medicine, University of California, San Francisco, CA, and investigator of the Alta and AFFINE studies. Our focus now is to confirm these exciting findings in the Phase 3 study, and to gather long-term data by following these patients and others in the Phase 3 study over a longer period of time.

Giroctocogene fitelparvovec was generally well tolerated. As previously reported, one patient in the 3 x 1013 vg/kg dose cohort had a treatment-related serious adverse event of hypotension (grade 3) and fever (grade 2), with symptoms of headache and tachycardia, which occurred six hours post-infusion with giroctocogene fitelparvovec, and which fully resolved within 24 hours. No other treatment-related serious adverse events were reported as of the cutoff date. Among the five patients in the 3 x 1013 vg/kg dose cohort, four received corticosteroids for liver enzyme (alanine aminotransferase, ALT) elevations. Three patients had subsequent ALT elevations that responded to corticosteroids. All episodes of ALT elevations fully resolved with oral corticosteroids, and as of the cutoff date no participants were on corticosteroids and no corticosteroid use has been initiated after week 52.

We continue to be encouraged by the findings from this Phase 1/2 study, which now include durable factor VIII expression through one year of follow-up, and we look forward to continuing to follow these patients, said Seng Cheng, Senior Vice President and Chief Scientific Officer of Pfizers Rare Disease Research Unit. With the first patient dosed in the Phase 3 AFFINE study in October 2020, we are on track for a readout from this pivotal Phase 3 trial in 2022, which will allow us to better assess the potential of our gene therapy across a larger sample size.

These latest results demonstrate that this gene therapy may bring clinical benefit to patients and has the potential to serve as an alternative to the burdensome standard of care for patients with hemophilia A, said Bettina Cockroft, M.D., M.B.A, Chief Medical Officer of Sangamo. We look forward to continuing to support our collaboration partners at Pfizer as they conduct the Phase 3 AFFINE study and assess the full potential of this promising therapy.

Pfizer and Sangamo plan to present further follow-up data from the Alta study when all five patients in the 3 x 1013 vg/kg dose cohort have been followed for at least two years.

About the Alta study

The Phase 1/2 Alta study is an open-label, dose-ranging, multicenter clinical trial designed to assess the safety and tolerability of giroctocogene fitelparvovec in patients with severe hemophilia A. The mean age of the 11 male patients assessed across four dose cohorts (9x1011 vg/kg - 2 patients, 2 x 1012 vg/kg - 2 patients, 1x1013 vg/kg - 2 patients and 3 x 1013 vg/kg - 5 patients) is 30 years (range 18-47 years). After one year of follow-up for all patients in the study, participants will be assessed every 6 months until they enroll into a long-term follow-up study.

About the AFFINE study

The Phase 3 AFFINE (NCT04370054) study is an open-label, multicenter, single arm study to evaluate the efficacy and safety of a single infusion of giroctocogene fitelparvovec in more than 60 adult (ages 18-64 years) male participants with moderately severe to severe hemophilia A. Eligible study participants will have completed at least six months of routine FVIII prophylaxis therapy during the lead-in Phase 3 study (NCT03587116) in order to collect pretreatment data for efficacy and selected safety parameters.

The primary endpoint is impact on annualized bleeding rate (ABR) through 12 months following treatment with giroctocogene fitelparvovec. This will be compared to ABR on prior FVIII prophylaxis replacement therapy. The secondary endpoints include FVIII activity level after the onset of steady state and through 12 months following infusion of giroctocogene fitelparvovec.

About giroctocogene fitelparvovec

The U.S. Food and Drug Administration has granted Orphan Drug, Fast Track, and regenerative medicine advanced therapy (RMAT) designations to giroctocogene fitelparvovec, which also received Orphan Medicinal Product designation from the European Medicines Agency. Giroctocogene fitelparvovec is being developed as part of a collaboration agreement for the global development and commercialization of gene therapies for hemophilia A between Sangamo and Pfizer. In late 2019, Sangamo transferred the manufacturing technology and the Investigational New Drug (IND) application to Pfizer.

About Hemophilia A

Hemophilia is a genetic hematological rare disease that results in a deficiency of a protein that is required for normal blood clotting clotting factor VIII in hemophilia A. The severity of hemophilia that a person has is determined by the amount of factor in the blood. The lower the amount of the factor, the more likely it is that bleeding will occur which can lead to serious health problems.

Hemophilia A occurs in approximately one in every 5,000-10,000 male births worldwide. For people who live with hemophilia A, there is an increased risk of spontaneous bleeding as well as bleeding following injuries or surgery. It is a lifelong disease that requires constant monitoring and therapy.

About Pfizer Rare Disease

Rare diseases include some of the most serious of all illnesses and impact millions of patients worldwide, representing an opportunity to apply our knowledge and expertise to help make a significant impact on addressing unmet medical needs. The Pfizer focus on rare disease builds on more than two decades of experience, a dedicated research unit focusing on rare disease, and a global portfolio of multiple medicines within a number of disease areas of focus, including rare hematologic, neurologic, cardiac and inherited metabolic disorders.

Pfizer Rare Disease combines pioneering science and deep understanding of how diseases work with insights from innovative strategic collaborations with academic researchers, patients, and other companies to deliver transformative treatments and solutions. We innovate every day leveraging our global footprint to accelerate the development and delivery of groundbreaking medicines and the hope of cures.

Click here to learn more about our Rare Disease portfolio and how we empower patients, engage communities in our clinical development programs, and support programs that heighten disease awareness.

Pfizer Inc.: Breakthroughs that change patients lives

At Pfizer, we apply science and our global resources to bring therapies to people that extend and significantly improve their lives. We strive to set the standard for quality, safety and value in the discovery, development and manufacture of health care products, including innovative medicines and vaccines. Every day, Pfizer colleagues work across developed and emerging markets to advance wellness, prevention, treatments and cures that challenge the most feared diseases of our time. Consistent with our responsibility as one of the world's premier innovative biopharmaceutical companies, we collaborate with health care providers, governments and local communities to support and expand access to reliable, affordable health care around the world. For more than 150 years, we have worked to make a difference for all who rely on us. We routinely post information that may be important to investors on our website at http://www.pfizer.com. In addition, to learn more, please visit us on http://www.pfizer.com and follow us on Twitter at @Pfizer and @Pfizer_News, LinkedIn, YouTube and like us on Facebook at http://www.facebook.com/Pfizer.

About Sangamo Therapeutics

Sangamo Therapeutics is committed to translating ground-breaking science into genomic medicines with the potential to transform patients lives using gene therapy, ex vivo gene-edited cell therapy, and in vivo genome editing and genome regulation. For more information about Sangamo, visit http://www.sangamo.com.

PFIZER DISCLOSURE NOTICE:

The information contained in this release is as of December 7, 2020. Pfizer assumes no obligation to update forward-looking statements contained in this release as the result of new information or future events or developments.

This release contains forward-looking information about an investigational hemophilia A therapy, giroctocogene fitelparvovec (SB-525, or PF-07055480), including its potential benefits and the anticipated timing and readout of the phase 3 clinical trial, that involves substantial risks and uncertainties that could cause actual results to differ materially from those expressed or implied by such statements. Risks and uncertainties include, among other things, the uncertainties inherent in research and development, including the ability to meet anticipated clinical endpoints, commencement and/or completion dates for our clinical trials, regulatory submission dates, regulatory approval dates and/or launch dates, as well as the possibility of unfavorable new clinical data and further analyses of existing clinical data; risks associated with interim data; the risk that clinical trial data are subject to differing interpretations and assessments by regulatory authorities; whether regulatory authorities will be satisfied with the design of and results from our clinical studies; whether and when drug applications for any potential indications for giroctocogene fitelparvovec may be filed in any jurisdictions; whether and when regulatory authorities in any jurisdictions may approve any such applications, which will depend on myriad factors, including making a determination as to whether the product's benefits outweigh its known risks and determination of the product's efficacy and, if approved, whether giroctocogene fitelparvovec will be commercially successful; decisions by regulatory authorities impacting labeling, manufacturing processes, safety and/or other matters that could affect the availability or commercial potential of giroctocogene fitelparvovec; uncertainties regarding the impact of COVID-19 on Pfizers business, operations and financial results; and competitive developments.

A further description of risks and uncertainties can be found in Pfizer's Annual Report on Form 10-K for the fiscal year ended December 31, 2019 and in its subsequent reports on Form 10-Q, including in the sections thereof captioned "Risk Factors" and "Forward-Looking Information and Factors That May Affect Future Results", as well as in its subsequent reports on Form 8-K, all of which are filed with the U.S. Securities and Exchange Commission and available at http://www.sec.gov and http://www.pfizer.com.

SANGAMO DISCLOSURE NOTICE:

This press release contains forward-looking statements regarding Sangamo's current expectations. These forward-looking statements include, without limitation, statements relating to the therapeutic potential of giroctocogene fitelparvovec (SB-525), including its potential clinical benefit to patients with hemophilia A and its potential as an alternative to the standard of care for patients with hemophilia A, the anticipated readout from the Phase 3 AFFINE study and the expected timing thereof, the plan and related timelines for sharing additional clinical data and other statements that are not historical fact. These statements are not guarantees of future performance and are subject to risks and uncertainties that are difficult to predict. Sangamos actual results may differ materially and adversely from those expressed. There can be no assurance that Sangamo will earn any additional milestone or royalty payments under the Pfizer collaboration. Factors that could cause actual results to differ include, but are not limited to, risks and uncertainties related to: the evolving COVID-19 pandemic and its impact on the global business environment, healthcare systems and the business and operations of Sangamo and Pfizer; the research and development process; the uncertain timing and unpredictable nature of clinical trial results, including the risks that therapeutic effects observed in clinical trial results will not be durable in patients and that final clinical trial data will not validate the safety and efficacy of giroctocogene fitelparvovec; reliance on results of early clinical trials, such as the Phase 1/2 Alta study, which results are not necessarily predictive of future clinical trial results, including the results in the Phase 3 AFFINE study; the unpredictable regulatory approval process for product candidates across multiple regulatory authorities; the manufacturing of products and product candidates; the commercialization of approved products; the potential for technological developments that obviate technologies used by Sangamo and Pfizer in giroctocogene fitelparvovec; the potential for Pfizer to terminate the giroctocogene fitelparvovec program or to breach or terminate its collaboration agreement with Sangamo; and the potential for Sangamo to fail to realize its expected benefits of its collaboration with Pfizer, including the risk that Sangamo may not earn any additional milestone or royalty payments under its collaboration with Pfizer. These risks and uncertainties are described more fully in Sangamo's filings with the U.S. Securities and Exchange Commission, including its most recent Quarterly Report on Form 10-Q for the quarter ended September 30, 2020. The information contained in this release is as of December 7, 2020, and Sangamo undertakes no duty to update forward-looking statements contained in this release except as required by applicable laws.

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Pfizer and Sangamo Announce Updated Phase 1/2 Results Showing Sustained Factor VIII Activity Levels in 3x1013 VG/KG Cohort Through One Year Following...