Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin's lymphoma.

BACKGROUND: Animal and clinical studies with plant-produced single-chain variable fragment lymphoma vaccines have demonstrated specific immunogenicity and safety. However, the expression levels of such fragments were highly variable and required complex engineering of the linkers. Moreover, the downstream processing could not be built around standard methods like protein A affinity capture. DESIGN: We report a novel vaccine manufacturing process, magnifection, devoid of the above-mentioned shortcomings and allowing consistent and efficient expression in plants of whole immunoglobulins (Igs). RESULTS: Full idiotype (Id)-containing IgG molecules of 20 lymphoma patients and 2 mouse lymphoma models were expressed at levels between 0.5 and 4.8 g/kg of leaf biomass. Protein A affinity capture purification yielded antigens of pharmaceutical purity. Several patient Igs produced in plants showed specific cross-reactivity with sera derived from the same patients immunized with hybridoma-produced Id vaccine. Mice vaccinated with plant- or hybridoma-produced Igs showed comparable protection levels in tumor challenge studies. CONCLUSIONS: This manufacturing process is reliable and robust, the manufacturing time from biopsy to vaccine is <12 weeks and the expression and purification of antigens require only 2 weeks. The process is also broadly applicable for manufacturing monoclonal antibodies in plants, providing 50- to 1000-fold higher yields than alternative plant expression methods.

Magnifection--a new platform for expressing recombinant vaccines in plants.

Today, plant biotechnology relies on two processes for delivery and expression of heterologous genes in plants: stable genetic transformation and transient infection with viral vectors. Although much faster, the transient route until recently was limited because of virus' low infectivity and its inability to carry average-size or larger transgenes. A recently developed new generation transfection technology overcomes these limitations by relying on Agrobacterium as an infective systemic agent that delivers viral replicons. This improved process is being used to simultaneously start transient gene amplification and high-level expression in all mature leaves of a plant, and such a transfection can be done on an industrial scale. This eclectic technology, called 'magnifection', combines advantages of three biological systems: vector efficiency and efficient systemic DNA delivery of Agrobacterium, speed and expression level/yield of a plant RNA virus, as well as posttranslational capabilities and low production costs of a plant. The proposed process allows for industrial production that does not require genetic modification of plants, that is much faster than previous methods, and that is biologically safe. Numerous applications in the area of vaccine manufacturing are being discussed.

Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family.

Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator (En/Spm) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (dSpm) element with a phosphinothricin herbicide resistance (BAR) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable dSpm transpositions. Treatments for both positive (BAR) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which approximately 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

Extreme structural heterogeneity among the members of a maize retrotransposon family.

A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of approximately 1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.

Retrotransposon insertion into the maize waxy gene results in tissue-specific RNA processing.

We previously reported that three alleles of the maize waxy (wx) gene were alternatively spliced as a result of the insertion of retrotransposons into intronic sequences. In addition, inefficient splicing of element sequences with the surrounding intron produced wild-type transcripts that presumably were responsible for the observed residual gene expression in the endosperm. In this study, we report that one of these alleles, wxG, has a tissue-specific phenotype with 30-fold more WX enzymatic activity in pollen than in the endosperm. Quantification of wxG-encoded transcripts in pollen and the endosperm demonstrates that this difference can be accounted for by tissue-specific differences in RNA processing. Specifically, there is approximately 30-fold more correctly spliced RNA in pollen than in the endosperm. Based on an analogy to similar examples of tissue-specific alternative splicing in animal systems, we hypothesize that the tissue-specific phenotype of the wxG allele may reflect differences in the concentration of splicing factors in these tissues.

Changes in state of the Wx-m5 allele of maize are due to intragenic transposition of Ds.

The molecular basis for the unusual phenotype conditioned by the waxy(Wx)-m5 Ds allele has been elucidated. Unlike most Ds alleles, Wx-m5 is phenotypically wild-type in the absence of Ac. We find that the Wx-m5 gene contains a 2-kb Ds element at -470 relative to the start of Wx transcription, representing the most 5' insertion of any transposable element allele characterized to date in plants. Despite its wild type phenotype, Wx-m5 has reduced levels of Wx enzymatic activity indicating that Ds insertion influences Wx gene expression. In the presence of Ac, Wx-m5 kernels have sectors of null expression on a wild-type background and give rise to stable wx and unstable wx-m germinal derivatives. Seventeen of 20 derivatives examined are wx-m alleles and at least 15 of these appear to result from intragenic transposition of Ds from -470 to new sites within the Wx gene. Three wx-m alleles contain two Ds elements, one at -470 and a second in Wx coding sequences. Surprisingly, only 3 out of 20 derivatives are stable wx mutants and these have sustained gross rearrangements of Wx and flanking sequences. For most other maize transposable element alleles somatic sectors and germinal derivatives usually arise following element excision or deletions of element sequences. In contrast, element insertion following intragenic transposition is apparently responsible for most of the somatic sectors and germinal derivatives of Wx-m5.