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The Antisense Approach in Amyloid Light Chain Amyloidosis: Identification of Monoclonal Ig and Inhibition of Its Production by Antisense Oligonucleotides in In Vitro and In Vivo Models¹

Satoko Ohno^*, Mitsuru Yoshimoto^*, Saho Honda^*, Sae Miyachi^*, Tadao Ishida^*, Fumio Itoh^2,*, Takao Endo^*, Susumu Chiba and Kohzoh Imai^*

^* First Department of Internal Medicine and Department of Neurology, Sapporo Medical University School of Medicine, Sapporo, Japan

	Abstract

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Primary amyloid L chain (AL) amyloidosis is a plasma cell disorder in which depositions of AL cause progressive organ failure. The lack of effective therapies for this fatal disease prompts exploration of newer treatment avenues. We have investigated the application of antisense oligonucleotides (AS) for the inhibition of monoclonal Ig production. The monoclonal L chain was identified by using primers designed for amplifying the human Ig V (V) region. We demonstrated that AS against L chain complementarity-determining regions inhibited the production of L chain in vitro. RPMI 8226 myeloma cells injected in SCID mice developed s.c. tumors. RT-PCR analysis showed V mRNA expression in the tumors. In addition, the presence of human Ig in the sera of mice given injection of RPMI 8226 cells was confirmed by ELISA. Administration of AS inhibited the expression of V mRNA in the s.c. tumors and decreased the concentration of L chain in serum. Therefore, we have shown that it is possible to determine the sequence of V mRNA and design specific complementary oligonucleotides, suggesting that treatment with V antisense could represent a rational novel approach to improve treatment outcome in AL amyloidosis.

	Introduction

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Systemic amyloid L chain (AL) amyloidosis is a plasma cell disorder in which the deposition of L chain causes progressive organ failure. The median survival of patients with AL amyloidosis is only 13 mo from diagnosis (1). Because this aggressive disease is characterized by the deposition of Ig L chains produced by clonal plasma cells, therapeutic approaches have been based on the use of alkylating agents active against multiple myeloma (MM). However, the median survival is still only 17–18 mo (2), in contrast to 3 years in MM without AL amyloidosis (3).

The pathogenesis of fibril formation from Ig L chains in AL amyloidosis is unknown. Because the majority of AL amyloid fibrils contain N termini of V region with only small amounts of the C region (4, 5, 6), it is thought that the variable domain plays an important role in the formation of pathological fibrils. In most of the trials with alkylating agents, patients who experienced reductions in their serum or urine monoclonal proteins had significantly prolonged survival compared with nonresponders (7). Therefore, treatment suppressing the production of amyloid fibril precursor protein could improve the prognosis of AL amyloidosis.

Antisense oligonucleotides (AS), providing a rationally designed tool to manipulate expression of specific genes, have been widely used in various in vitro and in vivo models and explored for their potential as therapeutic agents for the treatment of viral infections, cancers, and inflammatory disorders (8, 9, 10, 11, 12, 13, 14, 15). Human clinical trials using AS delivered by i.v. infusion or intravitreal injection are currently being conducted (16, 17). An AS approach that reduces the serum level of the amyloid fibril precursor protein could result in a major regression of the deposits.

In this study, we successfully determined nucleotide sequences of the human Ig V (V) region by RT-PCR and identified the monoclonal Ig that was produced by the neoplastic plasma cell clone. Based on the results obtained from V region sequences of each patient, we can design the AS that will specifically hybridize with the V region of mRNA. AS targeting V mRNA could inhibit the production of free -chain in a cell culture system. Furthermore, we have shown the reduction of human Ig production in SCID/RPMI 8226 mice by administration of AS against V mRNA. Our findings suggest that the use of V AS may be a novel approach to improve treatment outcome in AL amyloidosis.

	Materials and Methods

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Cells

The human MM cell line RPMI 8226 (IgG ; EBV^-) and U 266 (IgE ; EBV^-) were cultured in RPMI 1640 medium with 10% FBS.

PBMC and bone marrow mononuclear cells (BMMC) were obtained from three cases of MM, one case of AL amyloidosis with MM, and one case of monoclonal gammopathy of undetermined significance (MGUS) from which bone marrow aspirates were available. Table I summarized the clinicopathological data of the patients. Cells were isolated from whole blood or bone marrow aspirates using Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ).

C.B-17 SCID mice were obtained from Charles River Breeding Laboratories (Wilmington, MA) and thereafter were housed and maintained under specific pathogen-free conditions. RPMI 8226 cells were harvested, washed twice with PBS, and resuspended in PBS. Six-week-old SCID mice were injected s.c. with 1 x 10⁷ cells in 0.2 ml of PBS, pH 7.4, using a 25-gauge needle attached to a 1.0-ml syringe. Mice injected with PBS alone were used as negative controls. Tumor-bearing mice were monitored for up to 4 wk following emergence of palpable tumor.

RT-PCR

Total RNA was extracted from 1 x 10⁸ cells, PBMC and BMMC using TRIzol reagent (Life Technologies, Grand Island, NY) following the manufacturer’s instructions.

Universal oligonucleotide primers that Songsiviliai et al. (18) had designed for amplifying genes encoding the V region by PCR were adopted in this study. Upstream primers and downstream primers correspond to framework (FR)-1 and FR-4/J region, respectively.

First-strand cDNA was synthesized in a 20-µl reaction mixture containing 1 µg of total RNA, 5 mM MgCl₂, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1 mM each of NTP, 1 U/µl RNase inhibitor, 0.25 U/µl avian myeloblastosis virus reverse transcriptase (TaKaRa), and 2.5 µM random 9-mer primer. The tube was incubated at 30°C for 10 min, 42°C for 15 min, 99°C at 5 min and then cooled.

An 80-µl mixture containing 20 µl of the first-strand synthesis reaction mixture (cDNA-RNA hybrid), 0.2 µM each of the sense and antisense primers, 2.5 mM MgCl₂, 10 mM Tris-HCl, pH 8.3, 50 mM KCl, and 0.5 µl of Taq thermostable DNA polymerase (TaKaRa), was overlaid with paraffin oil and subjected to 30 cycles of PCR amplification using a thermal cycler. Each cycle consisted of 1 min at 94°C (denaturation), 1 min at 56°C (annealing), and 2 min at 72°C (extension).

Subcloning and sequencing of amplified DNA

The amplified DNA fragments were separated by electrophoresis on 2% low melting point agarose gel. After a distinct band of expected size was obtained, primer dimers were removed by using SUPREC-02 (TaKaRa). The purified PCR product was cloned into pGEM-T Easy Vectors (Promega, Madison, WI).

Nucleotide sequencing was accomplished with a Thermo Sequence II dye terminator cycle sequencing kit (Pharmacia Biotech) following the manufacturer’s instructions. Extension products were run for 16 h in an ABI 373S automated sequencer (PerkinElmer, Wellesley, MA).

Oligonucleotide and cell transfection

AS against complementarity-determining regions (CDR)-1, 2, 3 of the V regions of RPMI 8226 cells (designated AS (R) CDR-1, 2, 3) and the sense oligonucleotides (S (R) CDR-1, 2, 3) and scrambled AS containing the same nucleotide in random order (R (R) CDR-1, 2, 3) were synthesized (RPMI 8226 in Table II). In the same manner in U266 cells, antisense oligonucleotides (designated AS (U) CDR-1, 2, 3), sense oligonucleotides (designated S (U) CDR-1, 2, 3), and scrambled AS in random order (designated R (U) CDR-1, 2, 3) were synthesized. All base pairs were phosphorothioated (U266 in Table II).

Oligonucleotide administration

The AS against the V CDR-2 region of RPMI 8226 cells (AS (R) CDR-2) and corresponding sense oligonucleotide (S (R) CDR-2) were dissolved in physiological saline (0.9% NaCl) at a concentration of 2.0 mg/ml.

Four wk after s.c. injection of RPMI 8226 cells, the mice developed palpable tumors and were randomly assigned to three treatment groups; AS group (n = 10), sense oligonucleotide group (n = 10), and saline control group (n = 10). 7.5 mg/kg of oligonucleotide or saline was injected into the tumors daily for five consecutive days. Five mice were sacrificed 24 h after the final treatment. Total RNA was extracted from the tumors and the expression of V mRNA was assessed by RT-PCR, and the level of V in the serum was measured. The remaining five mice in each group were sacrificed 21 days after the final treatment. The expression of V mRNA in the s.c. tumors was assessed by RT-PCR, and the level of V in the serum was measured.

ELISA for V chain

Microtiter plates (96-well) were coated overnight with a 1/500 dilution of rabbit anti-human free -chain serum (DAKO A0101; Carpinteria, CA) in PBS (150 µl/well) at 4°C. Plates were then blocked with 3% BSA PBS. Duplicate serial dilutions of samples (150 µl/well) and pooled fresh medium as a control, were incubated for 2 h at 37°C. After washing, HRP-conjugated anti-human L chain Ab (diluted 1/500, 100 µl/well) (DAKO P0130) was added and incubated for 1 h at 37°C. After the final wash, o-phenylenediamine solution containing 3% H₂O₂ was added. Absorbance at 492 nm was determined on an ELISA reader (Easy Rrader EAR400; SRT-Labinstruments, Austria).

Statistical analysis

Data are expressed as mean ± SD. Statistical significance of differences was determined by the unpaired two-tailed Student’s t test. Differences were considered statistically significant for p < 0.01.

	Results

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Amplification of V mRNA in RPMI 8226 and U266 cells

An amplified DNA band of the expected size for V (330 bp) was detected in RPMI 8226 and U266, MM cell lines, but was not found in MKN45, a gastric cancer cell line (Fig. 1A, 1). The sequences of the PCR product of RPMI 8226 and U266 are shown in Fig. 1A, 2 and 3). By comparison with known sequences (http://www.ncbi.nlm.gov/BLAST), the sequence from RPMI 8226 and U266 cells were 99.02 and 99.98% homologous to the published sequence of RPMI 8226 or U266 cell line, respectively. These results showed that the primers used were adequate for amplifying genes encoding the V region.

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Agarose gel electrophoresis of the amplified cDNA from V regions of RPMI 8226 and U266 cell line (A1) and MM and/or AL amyloidosis patients (B1). Total RNA was isolated from 1 x 108 RPMI 8226 cells (R), U266 cells (U), MKN 45 cells (M), and PBMC (PB) or BMMC (BM) of the patients, and was reverse-transcribed to cDNA, which was used for the PCR amplification with universal oligonucleotide primers. The PCR product was subjected to agarose gel electrophoresis together with an appropriate m.w. maker, indicating that a DNA fragment of 330 bp was amplified. A2, Deduced amino acid sequences of the V region and AS target sites on V mRNA of the RPMI 8226 L chain. A3, Deduced amino acid sequences of the V region and AS target sites on V mRNA of the U266 L chain. The cDNA sequence and deduced amino acid sequences of the RPMI 8226 (A2) or U266 (A3) V region. The position to which the AS hybridize on the V mRNA are underlined. B2, Part of the primary structures of the V region of BMMC and PBMC in Case 1.

Amplification of V mRNA in MM, AL amyloidosis, and MGUS patients

A single band corresponding to the size expected for the V region (330 bp) was seen in both BMMC and PBMC of MM and AL amyloidosis patients (Fig. 1B, 1). Sequencing was conducted in 9–30 clones of cDNA fragment from each sample, which had been subcloned into the pGEM-T Easy vector. Table I summarizes the clinicopathological data and the number of identical clones in the five cases in this study. Part of the sequence of the V region of BMMC and PBMC in case 1 is represented in Fig. 1B, 2). In case 1 (a MM patient), in whom the percentage of plasma cells in the bone marrow (BM) was high (41.4%), seven of nine clones in the BM had an identical sequence. Similarly, five of nine clones in peripheral blood of the same patient had this sequence. In case 4 (the MM with AL amyloidosis patient), in whom the percentage of plasma cells in the BM was 10.0%, an identical V sequence was detected in 3 of 30 BM clones and in a single clone in the PBMC. These results suggest that identification of the monoclonal L chain could be possible in the cases, bearing over 10% plasma cells in the bone marrow.

Effect of AS on -chain production

Based on the sequence of RPMI 8226 and U266 V cDNA, 21-bp oligonucleotides were designed to hybridize to the CDR of V regions. Sense oligonucleotides and random oligonucleotides were included in this study as controls. The positions to which the AS hybridize on V mRNA are shown in Fig. 1A, 2 and 3; Table II depicts these sequences. The production of human free -chain for 24 h in the supernatant was determined by ELISA. The data are presented in Figs. 2 and 3. In RPMI 8226 cells, AS (AS (R) CDR-1, 2, 3) significantly decreased the free -chain production for 24 h. The random oligonucleotides (R (R) CDR-1, 2, 3), sense oligonucleotides (S (R) CDR-1, 2, 3), and AS for U266 cells (AS (U) CDR-1, 2, 3) did not inhibit -chain production. Also in U266 cells, AS specifically inhibited the -chain production. Treatment with any oligonucleotides (random, sense, antisense) had no effect on the proliferation of RPMI 8226 and U266 cells.

View larger version (56K): [in this window] [in a new window]   FIGURE 2. Inhibition of chain production by AS CDR-1 (A), AS CDR-2 (B), and AS CDR-3 (C) in RPMI 8226 cells. RPMI 8226 cells were treated with the indicated concentrations of oligonucleotides in the presence of 1.0 ml of serum-free RPMI 1640 medium, 20 µg of lipofectin (Life Technologies) and 50 µl of HEPES-buffered saline (150 mM NaCl and 20 mM HEPES, pH 7.4) for 5–6 h at 37°C. After incubation, the cells were washed with fresh medium and grown in 1.0 ml supplemented RPMI 1640 medium for 48 h. Human Ig levels in the medium were assayed by ELISA as described in Materials and Methods. The amount of free -chain production for 24 h is significantly decreased by AS(R) treatment (A and B). In CDR-2 and CDR-3, the data with random, sense, and AS(U) oligonucleotides are not shown (B). Data represent mean ± SD of nine independent determinations. *, Value significantly different from that obtained from control (p < 0.01).

View larger version (69K): [in this window] [in a new window]   FIGURE 3. Inhibition of -chain production by antisense in U266 cells.U266 cells were treated with oligonucleotides by the same method of RPMI 8226 cells. The amount of free -chain production for 24 h is significantly decreased by AS(U) treatment (A and B). In CDR-2 and CDR-3, the data with random, sense, and AS(R) oligonucleotides are not shown (B). Data represent mean ± SD of nine independent determinations. *, Value significantly different from that obtained from control (p < 0.01).

Reduction of V gene expression in the s.c. tumor of RPMI 8226 cells by AS

To determine whether AS is effective in vivo, tumor xenografts were developed by s.c. injection of RPMI 8226 cells into SCID mice and injected AS directly into the tumors. AS (R) CDR-2, which had shown the strongest inhibitory effect in vitro, wasselected for investigating in vivo effect. Primary tumors became palpable within 4 wk after injection, when oligonucleotide was administrated. Total RNA was extracted from fresh cell suspensions of the s.c. tumors of SCID mice at 1 and 21 days after administration of oligonucleotide. V mRNAs were found in the s.c. tumors. There was no difference between the saline control group and the sense-administered group in the expression of V mRNA. In contrast, the expression of the V mRNA was clearly decreased in tumors treated with AS (R) CDR-2 on the day 1-killed mice (Fig. 4A), and the suppression effect continued in the day 21-killed mice (Fig. 4B).

View larger version (28K): [in this window] [in a new window]   FIGURE 4. Effects of AS on the expression of human Ig mRNAs by RPMI 8226 xenografts in SCID mice. RT-PCR with universal oligonucleotide primers was performed on total RNA samples isolated from cells of SCID mice. SCID mice (n = 5 per group), injected s.c. with RPMI 8226 cells and saline (lanes 2 and 3), sense oligonucleotide (lanes 4 and 5), or AS (lanes 6 and 7). A dose of 7.5 mg/kg per day of oligonucleotides was administered. The mice were evaluated on day 1 (24 h after final treatment) (A) and day 21 (B). Lane 1, RPMI 8226 cell line cells; lanes 2–7, cells from the s.c. tumor (lanes 2 and 3, saline control; lanes 4 and 5, sense oligonucleotide administration; lanes 6 and 7, AS administration).

The amount of human L chain in sera of SCID mice was assessed by ELISA on 1 and 21 days after treatment. Human L chain was detected in the serum from mice s.c. injected with RPMI 8226 cells. Fig. 5 shows the concentration of human free -chain in mice sera treated with AS (R) CDR-2 on 1 and 21 days after the treatment. Neither sense oligonucleotide nor physiological saline had any inhibitory effects on the production of V. On day 1, the concentration of human free -chain in mice sera of the saline-, sense-, and antisense-administrated group was 8.38, 8.44, and 8.25 mg/L, respectively (Fig. 5A), which do not show statistical difference in those values. However on day 21, the concentration of human Ig in SCID/RPMI mice treated with AS (R) CDR-2 was significantly decreased compared with saline and sense control groups (Fig. 5B). Administration of AS had not inhibited the growing of tumors.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Effects of AS on the production of human Ig L chain in xenografted SCID mice. Human Ig L chain is detectable in the serum of MM-xenografted mice. SCID mice (n = 5 per group), injected s.c. with RPMI 8226 cells and saline (lane 2), sense oligonucleotide (lane 3), or AS (lane 4). A dose of 7.5 mg/kg per day of oligonucleotides was administered. Sera from SCID mice were analyzed by ELISA for human free L chain on days 1 (A) and 21 (B) after final treatment. Lane 1 is SCID mice not injected with RPMI 8226 cells. Data represent mean ± SD of nine independent determinations. *, The Student t test was used to calculate p values. The bars are means ± SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

AS can selectively block disease-causing genes, thereby inhibiting production of disease-associated proteins. Inhibition of gene expression by oligonucleotides occurs as the result of hybridization arrest (i.e., interference with the processing of mRNA by hybridization) and cleavage of the mRNA by RNase H (30, 31). CDR are Ag-binding loops and specific for the monoclonal Ig produced by the neoplastic plasma cell clone. Therefore, we expected that AS for CDR would not inhibit the normal Ig production.

It is possible that oligodeoxynucleotides give rise to unexpected cellular responses. Krieg et.al. (32, 33, 34, 35, 36) have revealed that oligodeoxynucleotides containing unmethylated CpG motifs could stimulate innate and adoptive immune responses. Such oligonucleotides could stimulate B cells to proliferate and secrete Ig and a variety of immune modulatory cytokines and other factors. Because RPMI 8226 cells and U266 cells are descendants of B cells, CpG containing oligonucleotides may exert some influence on the proliferation and -chain production of the cells. In control oligonucleotides (sense and random oligonucleotides), CpG sequences are identified within S (R) CDR-2, R (R) CDR-2, S (R) CDR-3, R (R) CDR-3, S (U) CDR-2, R (U) CDR-2, and R (U) CDR-3 oligonucleotides, but S (R) CDR-1, R (R) CDR-1, S (U) CDR-1, R (U) CDR-1, and S (U) CDR-3 contain no CG motif. After the treatment with these oligonucleotides, there was no significant difference between CpG oligo and non-CpG oligo in cell proliferation and chain production. For this reason, it is likely that the effect of CpG motif in oligonucleotides is negligible in our in vitro study.

RT-PCR analysis of AS-treated tumors showed a reduction of V mRNA levels; after 21 days of treatment, the effect of AS remained, however slight recovery of V mRNA was detected. Suppression of V mRNA expression in engrafts on day 1 would reflect on the reduction of concentration of free -chain on day 21. To maintain the AS effect, AS have to be administrated on a continuous basis, or the expression vector that express the AS continuously have to be adopted.

The purpose of the present study was to explore the potential use of AS in the treatment of AL amyloidosis. First, it was shown that the monoclonal Ig produced by the plasma cell clone could be easily identified by using RT-PCR, and then antisense oligonucleotides could be designed in each case. Second, the AS had a significant sequence-specific in vitro inhibitory effect on Ig production. Third, after s.c. administration of AS, significant inhibitory effects on Ig production were found in SCID mice with xenografts of human MM. Our study indicates the potential use of AS as a therapeutic agent for AL amyloidosis treatment. The major advantage of AS is to specifically inhibit Ig production while avoiding the serious side effects and much nonspecific toxicity caused by chemotherapeutic agents. In further studies, the route of administration and the problems relative to the pharmacokinetics and bioavailability properties of oligonucleotides need to be investigated.

	Acknowledgments

 
We thank Emiko Takeda for her skillful technical assistance.

	Footnotes

 
¹ This work was supported in part by grants from the Amyloidosis Research Committee, Surveys and Research on Specific Disease from the Ministry of Health, Labor and Welfare, Japan and Hokkaido Geriatrics Research Institute.

² Address correspondence and reprint requests to Dr. Fumio Itoh, First Department of Internal Medicine, Sapporo Medical University of Medicine, South-1, West-16, Chuo-ku, Sapporo, 060-8543, Japan. E-mail address: fitoh{at}sapmed.ac.jp

³ Abbreviations used in this paper: AL, amyloid L chain; MM, multiple myeloma; AS, antisense oligonucleotide; V, human Ig V; BM, bone marrow; BMMC, BM mononuclear cell; MGUS, monoclonal gammopathy of undetermined significance; FR, framework; CDR, complementarity-determining region.

Received for publication November 29, 2001. Accepted for publication July 24, 2002.

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