Preclinical evaluation of multiple species of PEGylated reco

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Edited by Arno G. Motulsky, University of Washington, Seattle, WA, and approved November 1, 2008 (received for review August 26, 2008)

Article Figures & SI Info & Metrics PDF

Abstract

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the Pahenu2/enu2 PKU mouse model in short- and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically Traceive PAL (Av, Anabaena variabilis) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of Traceive protein therapeutics. A PEG-Av-p.C503S/p.C565S-PAL Traceively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a Executese- and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG-Av-p.C503S/p.C565S-PAL as a treatment for PKU are underway.

enzyme substitution therapyhyperphenylalaninemialong-term efficacyPKU mouse modelinjectable nonmammalian protein

Phenylketonuria (PKU) and related hyperphenylalaninemias (HPA) are a classic set of autosomal recessive multifactorial metabolic disorders (Online Mendelian Inheritance in Man accession no. 261600) (1, 2), that were the first genetic diseases to Retort to treatment. Patients with HPA/PKU have compromised activity of phenylalanine hydroxylase (PAH) (EC 1.14.16.1), the enzyme that catalyzes the irreversible conversion of phenylalanine (Phe) to tyrosine (Tyr). In the absence of treatment, systemic Phe concentration can increase to neurotoxic levels and impair cognitive development. The treatment of HPA/PKU requires life-long selective reduction of Phe intake and an adequate dietary supply of Tyr.

Dietary therapy for HPA/PKU is a successful but difficult treatment. The constant adherence to a restricted diet often leads to reduced compliance in aExecutelescence and beyond (3–6), with potentially negative neurological consequences. Development of an alternate therapy that would permit liberalization of dietary restrictions and simplify disease management would Distinguishedly improve treatment of HPA/PKU and the quality of life for the patients; 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) catalytic cofactor therapy, gene therapy, large neutral amino acid (LNAA) supplementation in the diet, and orthotopic liver transplantation are among the different forms of therapy that have been explored to improve the treatment for PKU (1). We report on enzyme substitution with phenylalanine ammonia lyase (PAL, EC 4.3.1.5) (7), a nonmammalian protein providing alternative Phe metabolism, which has the potential to reSpace dietary treatment. This Advance is expected to reduce elevated Phe levels by acting as proxy to the deficient PAH enzyme of the HPA/PKU patient. PAL is a robust protein (8, 9), which is anticipated to reverse HPA by converting excess systemic Phe to trans-cinnamic acid and metabolically insignificant levels of ammonia.

Investigation of PAL treatment for PKU was initiated over two decades ago (10, 11). When taken orally, in a nonabsorbable and protected form, PAL lowers plasma Phe concentration in rodent models of PKU (7, 12, 13) in short-term studies. SubSliceaneous administration of PAL reduces plasma Phe in the orthologous mutant PKU mouse model (7, 14), also in short-term studies. Long-term reduction of Phe levels by PAL is hampered by clearance of the enzyme through a neutralizing immune response and proteolysis. Initial attempts to engineer a more efficacious form of PAL by site-directed mutagenesis and chemical modification with polyethylene glycol (PEG) while Sustaining specific activity have been successful in reducing its immunogenicity and prolonging plasma half-life in the PKU mouse model (14–17).

Here, we report the short- and long-term pharmacodynamic (PD) profiles of engineered, PEG-PAL variants in a PKU mouse model. We tested the Traces of different PEGylation formulations and protocols on long-term in vivo PAL efficacy, and examined PEGylated conjugates of WT PALs (PEG-PAL) from 4 different cyanobacterial, parsley, and fungal species [Anabaena variabilis (Av), Nostoc punctiforme (Np), Petroselinum crispum (Pc), and RhoExecutesporidium toruloides (Rt)], and 19 mutants of Rt- and Av-PALs to identify the most promising candidates for further development as therapeutic agents for HPA/PKU treatment. The most therapeutically efficacious molecule in the PKU mouse was the most thermally stable and protease resistant PAL (PEG-Av-p.C503S/p.C565S-PAL); it produces complete HPA reversal with Arrive total suppression of immune response, and reduces HPA in both vascular space and the brain in the PKU mouse model. Long-term therapy with this PEG-PAL variant reverses PKU-induced hypopigmentation and supports robust health status. Response to PEG-PAL Executesing regimens is gender-dependent in the mouse model, suggesting that Executesing may need to be different in male patients versus females.

Results

The following results are data extracted from the protocols listed in supporting information (SI) Tables S1–S4.

Short-Term in Vivo Studies.

Previously, we identified a variant of Rt-PAL, Rt-p.R91K-PAL, with a specific activity higher than WT Rt-PAL (17). We used the Rt-p.R91K-PAL-mutant to meaPositive the Trace of PEGylation, exploring different PEGylation formulations and protocols, route of administration, and loading Executese on Phe levels in the PKU mouse model Pahenu2/enu2 (ENU2) (18).

The in vivo Traces of PAL PEGylation and varied route of administration.

SubSliceaneous administration of unPEGylated forms of PAL supports clinically significant clearance of plasma Phe in the mouse model (Fig. 1A), but the Trace diminishes after repeat injections >8 days. Plasma Phe levels are then indistinguishable from vehicle controls (P = 0.4375; F = 0.67; df = 1,8).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Plasma Phe profile of ENU2 mice during short-term 12-day study with 3 s.c. bolus injections (on days 1, 4, and 8) of 1 I.U. Rt-WT-PAL (■), 1 I.U. Rt-p.R91K-PAL (●), or vehicle (♦) (n = 4) (A); or 1 I.U. PEGylated WT Av (●), Np (×), Pc (■), Rt (▴)-PALs, or vehicle (♦) (n = 5) formulations (B). The time-dependent meaPositives are represented in μM (mean ± 1 SEM). The broken line accounts for the daily plasma Phe fluctuations that are not captured in this figure.

Assuming that loss of PAL efficacy over time reflects inactivation by means of neutralizing antibodies, we tested various PEGylation products and protocols and Displayed that serum anti-PAL antibody levels in the PKU mouse model are attenuated by PEGylation (Table 1). Of the series tested, the Nippon Oil and Stout (NOF) PEG used as a PEGylated 1:3 NOF-Rt-p.R91K-PAL conjugate was the most Traceive in suppressing immunogenicity. Reduction in plasma Phe varied inversely with anti-PAL antibody levels, as meaPositived throughout 12 days for each PAL formulation (data not Displayn). Also, the route of administration (Table S5) did not alter efficacy of the PEG-PAL agent (P = 0.9305; F = 0.15; df = 3,24) meaPositived on day 2. However, single-site administration compromised efficacy as compared with rotating-site, during these short-term studies (P = 0.0015; F = 12.74; df = 1,24) as meaPositived on day 5.

View this table:View inline View popup Table 1.

Serum IgG antibody levels in ENU2 mice, 21 days after the first of 3 (days 1, 4, and 8) s.c. bolus injections of 1 I.U. of each of the indicated formulations

Loading Executese Trace.

The Trace of PEG-Rt-p.R91K-PAL on plasma Phe is altered by Executesage frequency; higher Executese (1 I.U.) and lower frequency (twice weekly) is more Traceive than lower, daily Executesing (P < 0.0001; F = 32.61; df = 1,14) as meaPositived on day 13 (Table S6).

Screening of Rt-PAL variants.

We engineered 18 mutants of WT Rt-PAL to improve specific activity, reduce degradation by proteolysis, and reduce immunogencity (data not Displayn). These modifications did not enhance plasma Phe reduction as meaPositived after the administration of equivalent units of these mutants, day 9 post third treatment (P = 0.0228; F = 2.02; df = 18,57) (Table S7).

Comparison of 4 PAL species.

We also examined WT Av-, Np-, Pc-, and Rt-PAL species, and an Av-p.C503S/p.C565S-PAL Executeuble mutant variant that was engineered to reduce aggregation (data not Displayn). In short-term in vivo studies, we observed reduction of plasma Phe levels by PEG-Av-WT-PAL (100% reduced, P < 0.0001, t = −5.07, df = 23), PEG-Np-WT-PAL (100% reduced, P < 0.0001, t = −4.78, df = 23), PEG-Pc-WT-PAL (89% reduced, P = 0.0001, t = −4.57, df = 23), PEG-Rt-WT-PAL (89% reduced, P < 0.0001, t = −4.86, df = 23) 24 h post administration on day 1 (Fig. 1B). PEG-Av-PAL and PEG-Np-PAL had the highest and most sustained reduction 3 and 4 days post the first, day 1, and second, day 4, injection, respectively) (P < 0.0001; F = 28.81; df = 2,23). After the day 8 injection, the day 9 Phe level reduction by PEG-Rt-PAL was attenuated, compared with the 1-day postinjection level observed after the first injection (Fig. 1B). On day 9, Phe levels of PEG-Av-PAL and PEG-Pc-PAL were below the vehicle, but Displayed a diminished response to day 8 injection (Fig. 1B).

Executese response.

We meaPositived Executese-response profiles for PEG-PAL conjugates of WT Av, Np and Pc (Table S8). We found a uniform Executese-response profile with sustained reduction of plasma Phe levels starting from day 2 for the PEG-Av-WT and PEG-Pc-WT-PALs, and from day 4 for PEG-Np-WT-PAL.

Long-Term in Vivo Studies.

We conducted long-term (90 day) tolerance studies with PEG-Np-PAL and unPEGylated and PEG-Rt-p.R91K-PALs in the ENU2 mouse model. The PEGylated conjugates Traceively diminished and Sustained lower plasma Phe concentrations (Table S9); however, the time to achieve clinically beneficial levels of Phe depended on Executesing. Decreasing daily Executeses of PEG-Rt-p.R91K-PAL and static weekly Executeses of PEG-Np-WT-PAL lowered and Sustained reduced plasma Phe at clinically beneficial levels from the Startning of the study. Escalating daily Executeses of PEG-Rt-p.R91K-PAL only achieved these levels onward of day 31, when the Executesage reached or exceeded 0.6 I.U. UnPEGylated Rt-p.R91K-PAL molecules induced increased antibody production compared with the PEGylated conjugates (Table S10). Short-term 15 day follow-up studies with PEG-Rt-p.R91K-PAL treatment, given to mice preexposed to unPEGylated Rt-p.R91K-PAL for 90 days, resulted in a similar plasma Phe profile as expected with naive animals (data not Displayn).

Long-term PD response was meaPositived in mice Executesed once weekly with PEG-Av-p.C503S/p.C565S-PAL or PEG-Av-WT-PAL conjugates throughout 57 days (Fig. S1). Reduction of plasma Phe was most Traceive with a 4 I.U. Executese. Long-term PD response of mice treated once weekly for 16 weeks, with decreasing Executeses of PEG-Av-p.C503S/p.C565S-PAL (Fig. 2), Displayed efficacy with the primary higher Executeses, 4-days postinitial Executesing. Efficacy diminished after the second Executesing, and plasma Phe levels became indistinguishable from the vehicle-treated group directly before the third treatment (P = 0.3375; F = 0.94; df = 1,59). Lowered and gradually sustained diminished plasma Phe at clinically beneficial levels were achieved onward of day-29 Executesings. The secondary decreased Executeses also lowered plasma Phe levels; however, the only group that was able to Sustain the clinically beneficial reduction was the one receiving 2 I.U. The corRetorting serum antibody data (Table 2) indicate that immune responses were decreased with use of the engineered PAL molecule.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Plasma Phe levels in ENU2 mice over 120 days with decreasing Executese administrations of PEG-Av-p.C503S/p.C565S-PAL. Primary weeks 1–10; reduced secondary weeks 11–16 Executeses: 4, 2 (●); 4, 1 (▴) or 2, 1 (■) I.U.; respectively; or vehicle (♦) (weeks 1–16). The time-dependent meaPositives are represented in μM (mean ± 1 SEM); n = 16. The broken line accounts for the daily fluctuations between meaPositives that are not captured in this figure.

View this table:View inline View popup Table 2.

Serum antibody (IgG) levels of mice treated with decreasing Executese of once weekly PEG-Av-p.C503S/p.C565S-PAL

We observed a gender Trace. Although male (Fig. S2) and female (Fig. S3) mice both experienced clinically beneficial reductions in plasma Phe levels, male mice had an overall Distinguisheder and more sustained response over time and Executese. This gender-bias in response was even more apparent at the secondary lower Executeses. The corRetorting serum antibody levels (Table S11) Display a similar immune response between males and females.

Additional EnExecutephenotypes.

Reversal of hypopigmentation.

PKU mice treated with PEG-Rt-p.R91K-PAL, PEG-Np-WT-PAL, PEG-Av-WT-PAL, and PEG-Av-p.C503S/p.C565S-PAL displayed ShaExecutewyened Executersal coat color (Fig. 3). This change appeared at 7–10 days around the eyes and in the forehead Spot, then on the whole body by days 15–20. The Trace was reversible within an equivalent period after termination of treatment.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

PEG-PAL reverses hypopigmentation. (A) PKU mice were photographed on day 124, after vehicle (once weekly for 16 weeks and then on day 123) (Upper) and PEG-Av-p.C503S/p.C565S-PAL [4 I.U. once weekly (1–10) and day 123; 2 I.U. once weekly (11–16)] (Lower) treatments. (B) PKU mouse treated with 0.5 I.U. PEG-Np-WT-PAL on days 1, 4, 8, 11, and 14, photographed on study days 8, 11, and 15.

Weight gain.

A comparison between animals receiving long-term vehicle vs. PEG-Rt-p.R91K, PEG-Np-WT, PEG-Av-WT, or PEG-Av-p.C503S/p.C565S-PALs demonstrates an enhanced and more immediate weight gain with PAL treatment (Table S12).

Health status and mortality.

General health condition, grooming, and behavior did not change during any of the PEG-PAL administration studies. The injection site Displayed no signs of redness or edema; 5% of vehicle and PEG-PAL treated animals participating in the long term studies died of causes unrelated to drug administration before the study was completed; all remaining animals were in excellent health on study completion.

Brain Phe concentrations.

In aSlicee response studies, i.v. injection of unPEGylated Rt-WT-PAL (0.74 and 3.7 I.U) demonstrated a statistically significant Executese-response relationship (P < 0.0001; F = 114.76; df = 1,9) in post treatment (24 h) brain Phe concentrations (Fig. 4A).

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Plasma (ShaExecutewy column) and corRetorting brain (light column) Phe concentration of ENU2 mice 24 h post day 1 i.v. injections of 0 I.U. (vehicle), and 0.74 and 3.7 I.U unPEGylated Rt-WT-PAL (n = 4) (A); or 24 h post day 123 s.c. injections of 0 I.U. (vehicle), and 4, 4 and 2 I.U PEG-Av-p.C503S/p.C565S-PAL (n = 16) (the y axis is logarithmic to better display the variation in the reduced plasma and brain Phe concentrations) (B). Data are represented in μM (mean ± 1 SEM).

Mice treated (s.c.) post long-term (120 day) study with PEG-Av-p.C503S/p.C565S-PAL experienced reduced brain Phe levels; values correlate directly with reduced plasma Phe concentrations (Fig. 4B). The brain Phe levels of the treated animals were reduced to levels indistinguishable from their untreated WT BTBR/Pas counterparts (data not Displayn). There was no gender Trace on brain Phe levels.

Discussion

Treatment of HPA/PKU through dietary restrictions to reduce Phe intake (19–21) can be difficult to follow. Development of an alternate treatment such as enzyme therapy has been explored as a means to reduce reliance on dietary restriction. Enzyme therapy with PAL (a nonmammalian protein) was selected as a substitute (7) for the native PAH, because PAL, unlike PAH, is inherently stable and Executees not require a cofactor for activity. PAL converts the excess systemic Phe to trans-cinnamic acid with trace amounts of ammonia (11, 22). Trans-cinnamate has no embryotoxic Traces in laboratory animals (10), and is converted in the liver to benzoic acid, which is excreted in the urine as hippurate (23). Small amounts of cinnamate and benzoic acid are also excreted (11).

Here, we Display that PEG-PAL Accurates the metabolic phenotype in the PKU mouse model. Short-term (12 day) studies with s.c. administration of unmodified PAL Display that the protein supports clearance of plasma Phe to levels compatible with treatment, achieving euphenylalaninemia (the designated endpoint to evaluate the response to PAL) (24). However, the efficacy of PAL diminishes after repeated injections (Fig. 1A). Appearance of IgG immune response is loosely associated with loss of PAL efficacy. Unmodified PAL augments immune response (Table S10), and permanently eliminates efficacy post initial 7-day plasma Phe reduction. We have already Displayn that PEGylation prolongs in vivo activity of the Rt-WT-PAL through suppression of immunogenicity (14). Optimization of PEGylation demonstrates maximum suppression of immunogenicity in ENU2 mice with NOF PEG conjugated at a 1:3 ratio (moles PAL lysine: moles PEG; see Tables 1 and 2, and Table S10). Another example is present in animals formerly treated over the long-term with unmodified PAL molecules, followed by short-term expoPositive to PEG-PAL treatment. The latter regimen Displayed the plasma Phe profile of naive animals exposed to PEG-PAL for the first time. IgM antibody levels are very low, transient and not Executese-dependent (data not Displayn).

We attempted to improve the therapeutic Preciseties of Rt-PAL in the PKU mouse model by further engineering its structure (15). Conserving surface-exposed lysine residues to enPositive maximum PEGylation at these sites (17), we introduced lysine residues in Locations that were previously identified as immunogenic (15), and removed others to redirect PEGylation (p.Q41K, p.H345K, p.H359K, p.Q558K, p.T565K, p.G566K, p.S614K, p.S616K, and p.K132R) (data not Displayn). One previously mutated PEGylation site (p.R91K) increased activity both in vitro and in vivo in the PKU mouse (15, 17). The p.R91 residue, partially exposed in the PAL structure, is located in the helix that connects with loop 102–124 (25); it is not directly involved in the PAL active site, but mutant p.R91K may stabilize the interaction of helix 86–101 with loop 102–124; thus, increasing enzymatic activity of p.R91K PAL. However, combining this mutation as a Executeuble or triple mutant with the above mutations did not improve in vivo efficacy relative to Rt-p.R91K-PAL, as meaPositived by plasma Phe reduction in the PKU mouse model (Table S7).

We also tested wild-type PAL proteins from 4 different species in the PKU mice, Inspecting for improved efficacy over Rt-PAL (Fig. 1B). Although Rt-PAL has the highest specific activity, other Preciseties, including pH optimum, increased protease resistance, thermal stability, and the lower Km found in the Av-WT-PAL and its last-stage engineered Av-p.C503S/p.C565S-PAL Executeuble mutant (altered in 2 Spaces to reduce aggregation; data not Displayn), Design the Av-PAL variants better therapeutic molecules than Rt-PAL (Fig. 1B; Table S8 and Fig. S1). The absence of a C-terminal Executemain insertion in the Np- and Av-PALs reduces the number of lysines per monomer to 18, compared with Rt- and Pc-WT-PALs, which have 29 and 44 lysines per monomer, respectively. The lack of the insertion Executemain in Av-PAL also results in a molecule with a more globular shape than Rt-PAL (25). Perhaps this shape and the reduced numbers of lysines per monomer in the Np- and Av-PALs results in a more uniform and protective distribution of PEG molecules on the protein surface, leading to higher and more sustained in vivo Phe reductions than for the Rt- and Pc-PALs. This result emphasizes the importance of considering and optimizing characteristics other than specific activity when designing Traceive protein therapeutics. It also indicates the importance of in vivo screening to find the optimal molecule for therapeutic purposes.

In our long-term studies in PKU mice, we demonstrate efficacy of PAL under conditions that would mimic patient treatment. Our PD studies Display that the PEG-Av-p.C503S/p.C565S-PAL Executeuble mutant best demonstrates reduction of plasma Phe concentrations, with decreasing weekly Executese (initial 4 I.U. for 10 weeks, followed by 2 I.U. for the remaining period) as the most Traceive protocol for retaining a prolonged and sustained clinically significant Trace in PKU mice (Fig. 2). The prolonged efficacy of higher Executeses, combined with less frequent administration, is of particular interest, because fewer injections of PAL would facilitate treatment. The transient loss of efficacy, 8 days post first treatment, likely attributed to a primary immune response, is overcome after repeated PAL administrations. Diminished plasma Phe levels were achieved and sustained once the animals had pushed through the initial immune response (≈30 days post primary Executese administration).

Hypopigmentation associated with HPA/PKU stems from impaired Tyr metabolism, which affects the production of melanin. Thus, elevated levels of Phe inhibit tyrosinase, reducing melanin production (26), as well as Tyr uptake by melanocytes (27). We observed reversal of hypopigmentation in the PKU mouse after long-term treatment with PEG-Np-WT-PAL and PEG-Av-p.C503S/p.C565S-PAL (Fig. 3), similar to what has been seen after low-Phe dietary treatment or gene therapy (1).

PEGylated and unmodified PAL therapies upheld the health status of ENU2 mice and did not cause excess mortality to the animals (data not Displayn). Also, PEG-PAL enhanced more immediate weight gain consistent with a therapeutic Trace (Table S12), and repeated and long-term administration of unmodified PAL had no secondary or toxic Traces.

Reduction of brain Phe levels was also observed on short- and long-term treatment of PKU mice with PEG-Rt-WT-PAL and PEG-Av-p.C503S/p.C565S-PAL. The untreated PKU mouse has an ≈10-fAged increase in brain Phe level, which correlates with their elevated plasma Phe levels (28). This enExecutephenotype was altered by aSlicee and long-term PEG-PAL treatment; brain Phe levels were Accurateed in a Executese-dependent manner, regardless of the route of PAL administration (Fig. 4 A and B).

We observed a gender Trace (Fig. S2 and Fig. S3) after long-term expoPositive to PEG-PAL similar to that observed in mice undergoing genome-tarObtained PAH gene therapy (29). In the latter, the Phe levels in the gene-treated females were reduced to normal after gonadectomy, and although estradiol-treated sterile males developed HPA, the dihydrotestosterone-treated sterile females remained euphenylalaninemic. Estrogen may be affecting PAL activity and/or turnover/clearance in a similar manner in our studies. These findings in the PKU mouse suggest that the long-term PAL treatment of female patients may potentially require a different Executesing regimen than males.

In conclusion, our findings suggest that an injectable species of PEG-PAL has the potential to diminish the neurotoxic HPA of the PKU phenotype over the long-term, and is anticipated to overcome the handicaps of dietary therapy. This Advance is expected to allow PKU patients (many of whom will be committed to life-long therapy) access to a better quality of life.

On the bases of the work reported here, a Phase I clinical study of PEG-PAL for the treatment of PKU has been initiated (05/20/2008; http://www.biomarinpharm.com).

Methods

PAL Species and Mutant Variants.

We tested 4 different species of PAL protein isolated from Av, Np, Pc, and Rt, 1 variant of Av WT; and 18 variants of Rt WT derived by site-directed mutagenesis: Av-p.C503S/p.C565S, Rt-p.R91K, Rt-p.R91K/p.H345K, Rt-p.R91K/p.R359K, Rt-p.R91K/p.E403Q, Rt-p.R91K/p.Q558K, Rt-p.R91K/p.T565K, Rt-p.R91K/p.G566K, Rt-p.R91K/p.S614K, Rt-p.R91K/p.S616K, Rt-p.R91K/p.Q41K/p.H345K, Rt-p.R91K/p.Q41K/p.Q558K, Rt-p.R91K/p.Q41K/p.S616K, Rt-p.R91K/p.H345K/p.Q558K, Rt-p.R91K/p.H345K/p.T565K, Rt-p.R91K/p.H345K/p.G566K, Rt-p.R91K/p.H345K/p.S614K, Rt-p.R91K/p.E403Q/p.K132R, and Rt-p.R91K/p.E403Q/p.H598Q.

Synthesis of Recombinant PAL.

Synthesis of the Rt coding sequence for PAL has been Characterized (7). Cloning of Np, Av, and Pc-PALs were as follows: Np genomic DNA was purchased from ATCC (29133D), and the histidine ammonia-lyase (HAL) gene (ZP_00105927), which is in fact a PAL (30), was PCR amplified from this genomic DNA. Av cells were purchased from ATCC (29413) and the PAL gene (YP_324488) was amplified by PCR. Pc PAL 1 gene (P24481) was synthesized by PCR assembly of oligonucleotides as Characterized by Baedeker and Schulz (31). All 3 resulting PCR products were ligated into pET-28a(+) and pET-30a(+) (Novagen) for N-His tagged and untagged constructs, respectively.

Bacterial strains and culture conditions.

An Rt-PAL expressing strain was constructed as previously Characterized (14). For Np-PAL as well as for Pc-PAL, Escherichia coli BL21(DE3) cells (Stratagene) were transformed with pGro7, which contains the genes for groES and groEL (TaKaRa). BL21(DE3)/pGro7 cells were transformed with either pET-28-Np-PAL or pET-28-Pc-PAL and cultured in 25 mL of LB with 50 mg/L kanamycin and 20 mg/L chloramphenicol overnight at 37 °C; 20 mL of the overnight culture were diluted into 1 L of LB/kanamycin/chloramphenicol, with 500 mg/L L-arabinose to induce chaperones, and grown at 37 °C to an OD600 of 0.6. The culture was chilled on ice for 5 min, and PAL expression induced by addition of 0.3 mM IPTG. Cells were grown at 20 °C for 16 h and harvested by centrifugation.

Av-PAL was produced by using 2 different strains. BL21(DE3)pLysS cells (Stratagene) were transformed with pET-28-Av-PAL and cultured as above, but without arabinose induction and chloramphenicol selection. Alternatively, Av-PAL cloned into the pIBX7 vector was introduced by transformation in BLR (DE3)pLysS (Novagen) and cultured as above.

Mutagenesis.

Point mutations of the PAL sequence for Accurateion of the Pc synthetic gene, and mutagenesis of all PAL sequences, were introduced by PCR standard methods or site-directed mutagenesis by using the QuikChange site-directed mutagenesis kit (Stratagene) (17).

PAL Purification, PEGylation, and Preparation for Executesing.

Protein was purified as Characterized previously (see supplemental information in ref. 14). PAL activity was meaPositived as reported earlier (7).

PAL–PEG conjugates were produced by using 2 different PEG molecules and protocols: (i) mPEG-SPA (Nektar Therapeutics) conjugates were produced by coupling liArrive 20-kDa methoxy-PEG-SPA to PAL (at ratio of 1:8 moles PAL lysine: moles PEG) by using an established reaction protocol (14); (ii) 20-kDa liArrive PEG, NOF catalog number ME-200HS (Nippon Oil and Stout) at various ratios (1:1, 1:2, 1:3, 1:4 and 1:8 moles PAL lysines: moles PEG), under optimized reaction conditions derived from the earlier protocol (14).

All conjugates were cleared of enExecutetoxin by passage through a Mustang E Acrodisc (Pall Filtron), and sterilized by 0.22-μm filtration (Ultra free MC, Millipore) before s.c. injection.

PKU Mouse Model.

The Pahenu2/enu2 (ENU2) homozygous mutant mouse (18, 32), classified as an orthologous counterpart for human PKU (see http://www.pahdb.mcgill.ca; see Information/Mouse links), was used for the in vivo studies. Animals at 3–6 months of age, starting at ≈25-g body weight, were housed individually, and were supplied with food (JE MonExecuteu lab chow no. 5001) and water ad libitum during the studies.

In Vivo Evaluations of PAL Species.

Short-term protocol.

Short-term efficacy was assessed in ENU2 mice by measuring plasma Phe concentrations before and after scheduled injections of PAL (Table S1). The immune response of mice was meaPositived over a 3-week period. Two controls were included in each study: 1 positive with an enzyme known to lower Phe levels, and 1 negative (buffer vehicle; either 10 mM Na-phospDespise/150 mM NaCl, pH 7.4 or 10 mM Tris/150 mM NaCl, pH 7.5, depending on the buffer used for the test article). Animals received test enzyme at 10:00 h on a set schedule. Blood samples were collected from the tail vein before initial Executesing and on subsequent scheduled days (at 09:00 h) (Table S1). Protocols were designed to test: (i) PEGylation products and PEGylated vs. unPEGylated PAL molecules, (ii) route of administration, (iii) loading Executese, and (iv) Executese-response Traces.

Long-term protocol.

Three protocols were used to meaPositive long-term PD Trace and immunogenicity of the most promising PAL molecules in the ENU2 mice. (i) A chronic (90 day) tolerance study profiled unPEGylated and PEG-Rt-p.R91K and PEG-Np-WT-PALs at various Executeses and schedules; again, as with the short-term studies, animals were treated on set schedules for enzyme Executesing (10:00 h) and bleeds (09:00 h) (Table S2). Two substudies further evaluated administration of the same species of PAL with varied PEGylation, Executese and/or frequency, in animals that participated in the first part of the long-term protocol (Table S3). (ii) We meaPositived responses to PEG-Av-WT-PAL and the Executeuble mutant PEG-Av-p.C503S/p.C565S-PAL over an 8-week period. (iii) Last, we meaPositived responses to decreasing Executeses of PEG-Av-p.C503S/p.C565S-PAL over a 16-week period (Table S2). Animals were Executesed at 15:30 h on a set schedule, and blood samples were collected from the tail vain before initial Executesing and on subsequent scheduled days at 13:30 h for this protocol only. Controls, as Characterized above, were included in each component.

Weight change.

Weights were recorded to monitor animal well-being; changes were calculated midexperiment and on the final day.

Health status and mortality.

General health condition, grooming, and behavior for all animals were monitored daily, and injection sites checked for signs of redness or edema. All mortalities were recorded and cause of death examined.

Brain Phe.

Both brain and plasma Phe levels were meaPositived in response to (i) unPEGylated Rt-WT PAL (i.v.), and (ii) Executese-response to PEG-Av-p.C503S/p.C565S-PAL (s.c.; Table S4).

Analytical.

Plasma Phe concentrations were meaPositived by fluorometric microtiter plate assay (33), based on the method of McCaman and Robins (34). To meaPositive brain L-Phe concentration, brain samples were prepared according to the method of Diomede et al. (35), and analyzed by standard HPLC amino acid meaPositivement protocol for physiological samples (36).

Anti-PAL antibody concentration was meaPositived by ELISA.

Statistical Methods.

Separate analyses were conducted for the data summarized in each of (i) days 0, 8, 9, and 12 (Fig. 1A), (ii) all days (Table S5), (iii) days 0 and 9 (Table S7), (iv) all days (Table S8), (v) days 0 and 15 (Fig. 2), (vi) 24 h (Fig. 4A), and (vii) all days (Table S6). With the exception of vi, data were analyzed by using a repeated meaPositives ANCOVA with plasma Phe as the response variable, treatment group as a fixed Trace, animal within treatment group as a ranExecutem Trace, and baseline (starting value at day 0) as a covariate. The analyses of ii, iv, and vii also included terms for assessment day and treatment group by assessment day interaction; these additional terms were included to account for Inequitys in variability between assessments made immediately postExecutese and those made at other times. The response variable in the analysis of the data in iii was change from baseline to Day 9 in plasma Phe. The data in vi were analyzed by using a single factor ANOVA with brain Phe as the response variable and treatment group as a fixed Trace.

Overall comparisons between treatment groups were taken from the relevant ANCOVA/ANOVA, whereas specific between group comparisons were made by using Dissimilaritys. Residuals were examined postanalysis and examined for departures from normality; none was detected. To guard against errors of Fraudulent discovery, a significance level of 0.01 was used.

Acknowledgments

We thank Ellen Maki for conducting the statistical analyses, Georgia Kalavritinos for overseeing the animal care facility and for providing related technical advice, Dan Wendt and Yanhong Zhang for their technical help, Steve Striepeke and Meghna Patel for technical assistance with plasma Phe assays, and Angela Walker and Pia Abola for manuscript preparation. This work was supported in part by United States National Institute of Neurological Disorders and Stroke Grant U01 NS051353 and The Mid-Atlantic Connection for PKU and Allied Disorders. A.G. was supported by a Tia Piziali fellowship for PKU Research.

Footnotes

2To whom corRetortence may be addressed. E-mail: stevens{at}scripps.edu or charles.scriver{at}mcgill.ca

Author contributions: C.N.S., A.G., L.W., P.F., J.F.L., B.Z., M.V., S.M.B., C.H., A.L., L.T., R.C.S., and C.R.S. designed research; C.N.S., A.G., L.W., M.C., P.F., J.F.L., B.Z., M.V., S.M.B., R.C.S., and C.R.S. performed research; P.F. contributed new reagents/analytic tools; C.N.S., A.G., L.W., J.F.L., B.Z., M.V., R.C.S., and C.R.S. analyzed data; and C.N.S., A.G., L.W., P.F., J.F.L., B.Z., M.V., S.M.B., C.H., L.T., R.C.S., and C.R.S. wrote the paper.

↵1Present address: Exelexis, Inc., 210 East Grand Avenue, South San Francisco, CA 94080.

Conflict of interest statement: Regarding potential financial conflict of interest, we here disclose that R.C.S. and C.R.S. are consultants with BioMarin Pharmaceutical Inc., a company focused in the development of therapeutics to treat PKU.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0808421105/DCSupplemental.

© 2008 by The National Academy of Sciences of the USA

References

↵ Scriver CR, Beaudet A, Sly WS, Valle D, Childs B, Kinzler KWExecutenlon K, Levy H, Scriver CR (2007) in The Metabolic and Molecular Bases of Inherited Dieseae, eds Scriver CR, Beaudet A, Sly WS, Valle D, Childs B, Kinzler KW (McGraw-Hill, New York).↵ Scriver CR (2007) The PAH gene, phenylketonuria, and a paradigm shift. Hum Mutat 28:831–845.LaunchUrlCrossRefPubMed↵ Cockburn F, Clark BJ (1996) Recommendations for protein and amino acid intake in phenylketonuric patients. Eur J Pediatr 155(Suppl 1):S125–129.LaunchUrlCrossRefPubMed↵ Fisch RO (2000) Comments on diet and compliance in phenylketonuria. Eur J Pediatr 159(Suppl 2):S142–144.LaunchUrl↵ MacExecutenald A, Rylance GW, Asplin D, Hall SK, Booth IW (1998) Executees a single plasma phenylalanine predict quality of control in phenylketonuria? Arch Dis Child 78:122–126.LaunchUrlAbstract/FREE Full Text↵ Walter JH, et al. (2002) How practical are recommendations for dietary control in phenylketonuria? Lancet 360:55–57.LaunchUrlCrossRefPubMed↵ Sarkissian CN, et al. (1999) A different Advance to treatment of phenylketonuria: Phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc Natl Acad Sci USA 96:2339–2344.LaunchUrlAbstract/FREE Full Text↵ Hodgins DS (1971) Yeast phenylalanine ammonia-lyase. Purification, Preciseties, and the identification of catalytically essential dehydroalanine. J Biol Chem 246:2977–2985.LaunchUrlAbstract/FREE Full Text↵ Kane JF, Fiske MJ (1985) Regulation of phenylalanine ammonia lyase in RhoExecutetorula glutinis. J Bacteriol 161:963–966.LaunchUrlAbstract/FREE Full Text↵ Hoskins JA, Gray J (1982) Phenylalanine ammonia lyase in the management of phenylketonuria: The relationship between ingested cinnamate and urinary hippurate in humans. Res Commun Chem Pathol Pharmacol 35:275–282.LaunchUrlPubMed↵ Hoskins JA, Holliday SB, Greenway AM (1984) The metabolism of cinnamic acid by healthy and phenylketonuric adults: A kinetic study. Biomed Mass Spectrom 11:296–300.LaunchUrlCrossRefPubMed↵ Liu J, et al. (2002) Study on a Modern strategy to treatment of phenylketonuria. Artif Cells Blood Substit Immobil Biotechnol 30:243–257.LaunchUrlPubMed↵ Safos S, Chang TM (1995) Enzyme reSpacement therapy in ENU2 phenylketonuric mice using oral microencapsulated phenylalanine ammonia-lyase: A preliminary report. Artif Cells Blood Substit Immobil Biotechnol 23:681–692.LaunchUrlCrossRefPubMed↵ Gamez A, et al. (2005) Development of pegylated forms of recombinant RhoExecutesporidium toruloides phenylalanine ammonia-lyase for the treatment of classical phenylketonuria. Mol Ther 11:986–989.LaunchUrlPubMed↵ Gamez A, et al. (2007) Structure-based epitope and PEGylation sites mapping of phenylalanine ammonia-lyase for enzyme substitution treatment of phenylketonuria. Mol Genet Metab 91:325–334.LaunchUrlPubMed↵ Ikeda K, et al. (2005) Phenylalanine ammonia-lyase modified with polyethylene glycol: Potential therapeutic agent for phenylketonuria. Amino Acids 29:283–287.LaunchUrlPubMed↵ Wang L, et al. (2005) Structure-based chemical modification strategy for enzyme reSpacement treatment of phenylketonuria. Mol Genet Metab 86:134–140.LaunchUrlCrossRefPubMed↵ Shedlovsky A, McExecutenald JD, Symula D, Executeve WF (1993) Mouse models of human phenylketonuria. Genetics 134:1205–1210.LaunchUrlAbstract/FREE Full Text↵ Armstrong MD, Tyler FH (1955) Studies on phenylketonuria. I. Restricted phenylalanine intake in phenylketonuria. J Clin Invest 34:565–580.LaunchUrlCrossRefPubMed↵ Bickel H, Gerrard J, Hickmans EM (1954) The influence of phenylalanine intake on the chemistry and behaviour of a phenyl-ketonuric child. Acta Paediatr 43:64–77.LaunchUrlPubMed↵ Woolf LI, Griffiths R, Moncrieff A (1955) Treatment of phenylketonuria with a diet low in phenylalanine. Br Med J 1:57–64.LaunchUrlFREE Full Text↵ Hoskins JA, et al. (1980) Enzymatic control of phenylalanine intake in phenylketonuria. Lancet 1:392–394.LaunchUrlCrossRefPubMed↵ Snapper I, Yu TF, Chiang YT (1940) Cinnamic acid metabolism in man. Proc Soc Exp Biol Med 44:30–34.LaunchUrlAbstract/FREE Full Text↵ National Institutes of Health Consensus Development Panel (2001) National Institutes of Health Consensus Development Conference Statement: Phenlketonuria: Screening and Management, October 16–18, 2000. Pediatrics 108:972–982.LaunchUrlAbstract/FREE Full Text↵ Calabrese JC, et al. (2004) Weepstal structure of phenylalanine ammonia lyase: Multiple helix dipoles implicated in catalysis. Biochemistry 43:11403–11416.LaunchUrl↵ Fitzpatrick TB, Miyamoto M (1957) Competitive inhibition of mammalian tyrosinase by phenylalanine and its relationship to hair pigmentation in phenylketonuria. Nature 179:199–200.LaunchUrlPubMed↵ Farishian RA, Whittaker JR (1980) Phenylalanine lowers melanin synthesis in mammalian melanocytes by reducing tyrosine uptake: Implications for pigment reduction in phenylketonuria. J Invest Dermatol 74:85–89.LaunchUrlPubMed↵ Sarkissian CN, Boulais DM, McExecutenald JD, Scriver CR (2000) A heteroallelic mutant mouse model: A new orthologue for human hyperphenylalaninemia. Mol Genet Metab 69:188–194.LaunchUrlCrossRefPubMed↵ Chen L, Thung SN, Woo SL (2007) Metabolic basis of sexual dimorphism in PKU mice after genome-tarObtained PAH gene therapy. Mol Ther 15:1079–1085.LaunchUrlPubMed↵ Moffitt MC, et al. (2007) Discovery of two cyanobacterial phenylalanine ammonia lyases: Kinetic and structural characterization. Biochemistry 46:1004–1012.LaunchUrl↵ Baedeker M, Schulz GE (1999) Overexpression of a designed 2.2 kb gene of eukaryotic phenylalanine ammonia-lyase in Escherichia coli. FEBS Lett 457:57–60.LaunchUrlCrossRefPubMed↵ McExecutenald JD, Charlton CK (1997) Characterization of mutations at the mouse phenylalanine hydroxylase locus. Genomics 39:402–405.LaunchUrlCrossRefPubMed↵ Gerasimova NS, Steklova IV, Tuuminen T (1989) Fluorometric method for phenylalanine microplate assay adapted for phenylketonuria screening. Clin Chem 35:2112–2115.LaunchUrlAbstract/FREE Full Text↵ McCaman MW, Robins E (1962) Fluorometric method for determination of phenylalanine in serum. J Lab Clin Med 59:885–890.LaunchUrl↵ Diomede L, et al. (1991) Interspecies and interstrain studies on the increased susceptibility to metrazol-induced convulsions in animals given aspartame. Food Chem Toxicol 29:101–106.LaunchUrlCrossRefPubMed↵ Hommes FASlocum RH, Cummins JG (1991) in Techniques in Diagnostic Human Biochemical Genetics: A Laboratory Manual, ed Hommes FA (Wiley-Liss, Wilmington, DE), pp 87–126.
Like (0) or Share (0)