Karin E. Lundin

Biological Activity and Biotechnological Aspects of Peptide Nucleic Acid

During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

Hypomorphic homozygous mutations in phosphoglucomutase 3 (PGM3) impair immunity and increase serum IgE levels

BACKGROUND Recurrent bacterial and fungal infections, eczema, and increased serum IgE levels characterize patients with the hyper-IgE syndrome (HIES). Known genetic causes for HIES are mutations in signal transducer and activator of transcription 3 (STAT3) and dedicator of cytokinesis 8 (DOCK8), which are involved in signal transduction pathways. However, glycosylation defects have not been described in patients with HIES. One crucial enzyme in the glycosylation pathway is phosphoglucomutase 3 (PGM3), which catalyzes a key step in the synthesis of uridine diphosphate N-acetylglucosamine, which is required for the biosynthesis of N-glycans. OBJECTIVE We sought to elucidate the genetic cause in patients with HIES who do not carry mutations in STAT3 or DOCK8. METHODS After establishing a linkage interval by means of SNPchip genotyping and homozygosity mapping in 2 families with HIES from Tunisia, mutational analysis was performed with selector-based, high-throughput sequencing. Protein expression was analyzed by means of Western blotting, and glycosylation was profiled by using mass spectrometry. RESULTS Mutational analysis of candidate genes in an 11.9-Mb linkage region on chromosome 6 shared by 2 multiplex families identified 2 homozygous mutations in PGM3 that segregated with disease status and followed recessive inheritance. The mutations predict amino acid changes in PGM3 (p.Glu340del and p.Leu83Ser). A third homozygous mutation (p.Asp502Tyr) and the p.Leu83Ser variant were identified in 2 other affected families, respectively. These hypomorphic mutations have an effect on the biosynthetic reactions involving uridine diphosphate N-acetylglucosamine. Glycomic analysis revealed an aberrant glycosylation pattern in leukocytes demonstrated by a reduced level of tri-antennary and tetra-antennary N-glycans. T-cell proliferation and differentiation were impaired in patients. Most patients had developmental delay, and many had psychomotor retardation. CONCLUSION Impairment of PGM3 function leads to a novel primary (inborn) error of development and immunity because biallelic hypomorphic mutations are associated with impaired glycosylation and a hyper-IgE-like phenotype.

Biological activity and biotechnological aspects of locked nucleic acids

Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). The use of LNA in antisense ONs, including gapmers, splice-switching ONs, and siLNA, as well as antigene ONs, is reviewed. Pharmacokinetics as well as pharmacodynamics of LNA ONs and a description of selected compounds in, or close to, clinical testing are described. In addition, new LNA modifications and the adaptation of enzymes for LNA incorporation are reviewed. Such enzymes may become important for the development of stabilized LNA-containing aptamers.

Zorro locked nucleic acid induces sequence-specific gene silencing

Locked nucleic acids (LNAs) are synthetic analogs of nucleic acids that contain a bridging methylene carbon between the 2′ and 4′ positions of the ribose ring. In this study, we generated a novel sequence‐specific antigene molecule “Zorro LNA”, which simultaneously binds to both strands, and that induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in a cellular context. By comparing the Zorro LNA with linear LNA as well as an optimized bisPNA (pep‐tide nucleic acid) oligonucleotide directed against the same target sites, respectively, we found that the Zorro LNA construct was unique in its ability to arrest gene transcription in mammalian cells. To our knowledge, this is the first time that in mammalian cells, gene transcription was blocked by a nucleic acid analog in a sequence‐specific way using low but saturated binding of a blocking agent. This offers a novel type of antigene drug that is easy to synthesize.—Ge R., Heinonen, J. E., Svahn, M. G., Mohamed, A. J., Lundin, K. E., Smith C. I. E. Zorro locked nucleic acid induces sequence‐specific gene silencing. FASEB J. 21, 1902–1914 (2007)

A synthetic snRNA m3G-CAP enhances nuclear delivery of exogenous proteins and nucleic acids

Accessing the nucleus through the surrounding membrane poses one of the major obstacles for therapeutic molecules large enough to be excluded due to nuclear pore size limits. In some therapeutic applications the large size of some nucleic acids, like plasmid DNA, hampers their access to the nuclear compartment. However, also for small oligonucleotides, achieving higher nuclear concentrations could be of great benefit. We report on the synthesis and possible applications of a natural RNA 5′-end nuclear localization signal composed of a 2,2,7-trimethylguanosine cap (m3G-CAP). The cap is found in the small nuclear RNAs that are constitutive part of the small nuclear ribonucleoprotein complexes involved in nuclear splicing. We demonstrate the use of the m3G signal as an adaptor that can be attached to different oligonucleotides, thereby conferring nuclear targeting capabilities with capacity to transport large-size cargo molecules. The synthetic capping of oligos interfering with splicing may have immediate clinical applications.

Gene Transfer to Mouse Heart and Skeletal Muscles Using a Minicircle Expressing Human Vascular Endothelial Growth Factor

Background: Gene transfer to heart muscle is a promising modality to treat ischemic heart disease. However, current vectors are inefficient and need to be improved. A novel vector system that shows great promise is the minicircle (MC) vector being smaller than conventional plasmid vectors and devoid of bacterial sequences. Aims: To study gene transfer of MC DNA, expressing the human vascular endothelial growth factor (hVEGF), to mouse heart and skeletal muscles and to compare it with one of the efficient plasmids used in cardiovascular trials, the phVEGF165 containing the same expression cassette as the MC. Results: The MC and the phVEGF165 plasmid show similar expression patterns both in vitro and in mouse heart and skeletal muscle studies in vivo on molar basis (equal expression in heart 24 hours, 0.9 fold lower expression from MC in heart and 1.9 fold higher in skeletal muscle at 7 days), whereas on weight basis the MC construct was more efficient in skeletal muscle (5.6 fold higher expression, P < 0.05), and at least as efficient in heart (1.6 fold higher expression). Conclusions: The gene expression is similar in the 2 vector systems, so the smaller size and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene therapy.

Peptides isolated from random peptide libraries on phage elicit a neutralizing anti-HIV-1 response: analysis of immunological mimicry.

Peptides binding to a murine, human immunodeficiency virus type 1 (HIV‐1) neutralizing monoclonal antibody (F58/H3) were isolated from two random peptide libraries expressed on the surface of phage. The antibody was originally elicited by immunization with HIV‐1 envelope protein gp120LAI, and has previously been shown to interact with the ‐I–GPGRA‐ motif of the V3 loop. The peptide libraries consisted of nine or 15 random amino acid residues flanked by two cysteines, and fused to the amino terminal end of the cpIII protein on the filamentous phage. Selection of specific peptides was carried out in three rounds, with decreasing antibody concentration. An expected peptide motif ‐GPGRA‐, a similar segment, ‐GPAR‐, and two unrelated motifs ‐FRLLG‐ and ‐WRM/ALG‐ were selected. Binding of antibody was tested both to synthetic peptides in solution, and the corresponding peptide on phage. The GPXR motifs bound in both formats, while the FRLLG bound antibody only when present on the phage. The reactivity of peptides on phage was highly dependent on an intact disulphide bond between the cysteines flanking the peptide. The molecular mimicry of the found motifs was tested by immunizing mice and rabbits with conjugated synthetic peptides or peptide on phage. In mice, peptide‐specific antisera were raised, but no reactivity to the whole protein (gp120) was detected. In rabbits, however, this was accomplished with the ‐GPGRA‐ containing peptide when present on phage. In addition, this antisera precipitated virus particles, and neutralized HIV‐1SF2 virus in vitro.

Adding functional entities to plasmids

Non‐viral gene therapy constitutes an alternative to the more common use of viral‐mediated gene transfer. Most gene transfer methods using naked DNA are based upon non‐sequence‐specific interactions between the nucleic acid and cationic lipids (lipoplex) or polymers (polyplex). We have developed a technology in which functional entities hybridize in a sequence‐specific manner to the nucleic acid (bioplex). This technology is still in its infancy, but has the potential to become a useful tool, since it allows the construction of highly defined complexes containing a variety of functional entities. In its present form the bioplex technology is based upon the use of peptide/nucleic acids (PNA) as anchors. Single, or multiple, functional entities are directly coupled to the anchors. By designing plasmids, or oligonucleotides, with the corresponding anchor target sequence, complexes with desired composition can easily be generated. The long‐term aim is to combine functional entities in order to achieve optimal, synergistic interactions allowing enhanced gene transfer in vivo. Copyright

Micro-minicircle Gene Therapy: Implications of Size on Fermentation, Complexation, Shearing Resistance, and Expression

The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, super-coiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.

A specific assay measuring binding of 125I-Gp 120 from HIV to T4+/CD4+ cells

The HIV (HTLV-III) envelope glycoprotein, Gp120, was isolated from virus-infected tissue culture cells using affinity chromatography. A radioimmunoassay was developed to determine the degree of iodinated Gp120 to target CD4+ (T4+) cells. 125I-Gp120 could be shown to selectively bind to CD4+ cells only. The Gp120 remained bound to these cells after repeated washes. Monoclonal anti-CD4 antibodies block the binding of Gp120 to CD4+ cells. Monoclonal antibodies to other cell surface components do not interfere with 125I-Gp120 binding. All IgG antibodies from HIV seropositive donors tested block 125I-Gp120 binding, though with variable titers. We believe that this assay provides further proof for the use of CD4 (T4) as a component of the receptor for HIV. It represents a safe, objective and sensitive method for the analysis of Gp120-CD4 interactions, as well as the potential of antibodies to interfere with this binding.