Development Of Vectors For Gene Therapy Research Articles

Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM.

In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method.

MRNA manufacturing platform using CIMmultus® and CIMac® technology for high recovery of stable mRNA drug substance with rapid HPLC analytics

<h3>Watch the video or read the poster to learn:</h3> <ul><li>Non-affinity chromatography with PrimaS provides purification for IVT processes with co-transcriptional polyadenylation</li><li>The Sartorius mRNA purification toolbox provides a scalable chromatographic solution for commercial manufacturing of mRNA therapeutics</li><li>CIMac analytical HPLC columns can resolve individual IVT reaction components in minutes, resolving the analytical bottleneck in optimization of IVT reaction conditions</li><li>Rapid HPLC analytics can improve in-process and quality control of the mRNA production process</li></ul><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Rok copy.jpg" data-image="1"></div><div class="author-det"> <b>Rok Sekirnik</b> graduated with a degree in chemistry from the University of Oxford, where he also completed his PhD. He has worked in the areas of mAb, vaccine and gene therapy vector development in various roles, including Principal Scientist at Novartis and Project Manager at Batavia Biosciences. His team at BIA Separations is focused on developing high-yielding, cost-effective purification approaches for pDNA and mRNA production. </div></div></div>

Development of Gene Therapy Vectors: Remaining Challenges.

Almost 20 years after the tragic death of a young patient due to an experimental gene therapy trial to treat Ornithine Transcarboxylase deficiency, the FDA approved its first landmark gene therapy drug i.e. Luxturna® to treat inherited blindness, and dozens of gene therapy studies are underway. Whether it is replacing the mutant copies of the gene with the wild type one or editing the mutant one in or ex-vivo to elicit the production of functional proteins, numerous viral and non-viral vectors for delivering the gene payload are being evaluated. While, non-viral vectors avoid or mitigate limiting factors such as immunogenicity and the presence of neutralizing antibodies (NAbs), viral vectors such as recombinant adeno-associated viruses (AAVs) have shown early success as a delivery vehicle, because of the overall safety, target specificity, and long-term stability profile. Nonetheless, multiple challenges during the AAV product development and approval process are still looming. AAV serotypes are continuously being engineered which requires multiple cell-based assays to not only assess the neutralizing antibodies (NAb) seroprevalence but also to develop the in-vitro bio potency assays. Hence, we focus on some critical aspects of the AAVs that determine the path forward for pre-clinical and clinical product development.

Novel Lung Tropic Adeno-Associated Virus Capsids for Therapeutic Gene Delivery.

Efforts to identify mutations that underlie inherited genetic diseases combined with strides in the development of gene therapy vectors over the last three decades have culminated in the approval of several adeno-associated virus (AAV)-based gene therapies. Genetic diseases that manifest in the lung such as cystic fibrosis (CF) and surfactant deficiencies, however, have so far proven to be elusive targets. Early clinical trials in CF using AAV serotype 2 (AAV2) achieved safety, but not efficacy endpoints; however, importantly, these studies provided critical information on barriers that need to be surmounted to translate AAV lung gene therapy toward clinical success. Bolstered with an improved understanding of AAV biology and more clinically relevant lung models, next-generation molecular biology and bioinformatics approaches have given rise to novel AAV capsid variants that offer improvements in transduction efficiency, immunological profile, and the ability to circumvent physical barriers in the lung such as mucus. This review discusses the principal limiting barriers to clinical success in lung gene therapy and focuses on novel engineered AAV capsid variants that have been developed to overcome those challenges.

Gene Therapy for Human Sensorineural Hearing Loss.

Hearing loss is the most common sensory impairment in humans and currently disables 466 million people across the world. Congenital deafness affects at least 1 in 500 newborns, and over 50% are hereditary in nature. To date, existing pharmacologic therapies for genetic and acquired etiologies of deafness are severely limited. With the advent of modern sequencing technologies, there is a vast compendium of growing genetic alterations that underlie human hearing loss, which can be targeted by therapeutics such as gene therapy. Recently, there has been tremendous progress in the development of gene therapy vectors to treat sensorineural hearing loss (SNHL) in animal models in vivo. Nevertheless, significant hurdles remain before such technologies can be translated toward clinical use. These include addressing the blood-labyrinth barrier, engineering more specific and effective delivery vehicles, improving surgical access, and validating novel targets. In this review, we both highlight recent progress and outline challenges associated with in vivo gene therapy for human SNHL.

CRISPR/Cas9-generated p47phox-deficient cell line for Chronic Granulomatous Disease gene therapy vector development

Development of gene therapy vectors requires cellular models reflecting the genetic background of a disease thus allowing for robust preclinical vector testing. For human p47phox-deficient chronic granulomatous disease (CGD) vector testing we generated a cellular model using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to introduce a GT-dinucleotide deletion (ΔGT) mutation in p47phox encoding NCF1 gene in the human acute myeloid leukemia PLB-985 cell line. CGD is a group of hereditary immunodeficiencies characterized by impaired respiratory burst activity in phagocytes due to a defective phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In Western countries autosomal-recessive p47phox-subunit deficiency represents the second largest CGD patient cohort with unique genetics, as the vast majority of p47phox CGD patients carries ΔGT deletion in exon two of the NCF1 gene. The established PLB-985 NCF1 ΔGT cell line reflects the most frequent form of p47phox-deficient CGD genetically and functionally. It can be differentiated to granulocytes efficiently, what creates an attractive alternative to currently used iPSC models for rapid testing of novel gene therapy approaches.

Production of canine adenovirus type 2 in serum-free suspension cultures of MDCK cells.

The potential of adherent Madin Darby Canine Kidney (MDCK) cells for the production of influenza viruses and canine adenovirus type 2 (CAV-2) for vaccines or gene therapy approaches has been shown. Recently, a new MDCK cell line (MDCK.SUS2) that was able to grow in suspension in a fully defined system was established. In this work, we investigated whether the new MDCK.SUS2 suspension cell line is suitable for the amplification of CAV-2 under serum-free culture conditions. Cell growth performance and CAV-2 production were evaluated in three serum-free media: AEM, SMIF8, and EXCELL MDCK. CAV-2 production in shake flasks was maximal when AEM medium was used, resulting in an amplification ratio of infectious particles (IP) of 142 IP out/IP in and volumetric and cell-specific productivities of 2.1 × 10(8) IP/mL and 482 IP/cell, respectively. CAV-2 production was further improved when cells were cultivated in a 0.5-L stirred tank bioreactor. To monitor infection and virus production, cells were analyzed by flow cytometry. A correlation between the side scatter measurement and CAV-2 productivity was found, which represents a key feature to determine the best harvesting time during process development of gene therapy vectors that do not express reporter genes. This work demonstrates that MDCK.SUS2 is a suitable cell substrate for CAV-2 production, constituting a step forward in developing a production process transferable to industrial scales. This could allow for the production of high CAV-2 titers either for vaccination or for gene therapy purposes.

A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17+)-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE.

Gene therapy as a cure for color blindness

Red‐green color blindness most often results from the absence of the long‐(L) or middle‐(M) wavelength sensitive visual photopigments. It is the most common single locus genetic disorder. Recent advances in the development of gene therapy vectors have offered the promise of gene therapy cures for inherited vision disorders; however, classical visual deprivation experiments show that for at least some features of the visual system, there is a “critical period” in development in which visual input is required for proper circuitry to form. Here, we performed subretinal injections of a gene therapy vector designed to introduce a third cone type in the retina of dichromatic (color blind) squirrel monkeys. The vector was adeno‐associated virus carrying the gene for a human L cone opsin under control of an L/M cone specific enhancer and promoter. The effects of the gene therapy treatment were monitored using color vision behavioral testing and electroretinography. Results indicate that trichromacy can arise from a single addition of a third cone class and it does not require an early developmental process. This provides a positive outlook for the potential of gene therapy to cure adult vision disorders and as a tool for exploring neural plasticity of the visual system.

Generation and Biological Properties of a Recombinant Dodecahedron Containing the Short Fiber Protein of the Human Adenovirus 41

Objective: In order to gain further insight into the function of the enteric adenovirus short fiber (SF), we have constructed a recombinant dodecahedron containing the SF protein of HAdV-41 and the HAdV-3 penton base. Methods: Recombinant baculoviruses expressing the HAdV-41 SF protein and HAdV-3 penton base were cloned and amplified in Sf9 insect cells. Recombinant dodecahedra were expressed by coinfection of High Five™ cells with both baculoviruses, 72 h post-infection. Cell lysate was centrifuged on sucrose density gradient and the purified recombinant dodecahedra were recovered. Results: Analysis by negative staining electron microscopy demonstrated that chimeric dodecahedra made of the HAdV-3 penton base and decorated with the HAdV-41 SF were successfully generated. Next, recombinant dodecahedra were digested with pepsin and analyzed by Western blot. A ‘site-specific’ proteolysis of the HAdV-41 SF was observed, while the HAdV-3 penton base core was completely digested. Conclusion: These results show that, in vitro, the HAdV-41 SF likely undergoes proteolysis in the gastrointestinal tract, its natural environment, which may facilitate the recognition of receptors in intestinal cells. The results obtained in the present study may be the basis for the development of gene therapy vectors towards the intestinal epithelium, as well as orally administered vaccine vectors, but also for the HAdV-41 SF partner identification.

Transport and egress of herpes simplex virus in neurons

The mechanisms of axonal transport of the alphaherpesviruses, HSV and pseudorabies virus (PrV), in neuronal axons are of fundamental interest, particularly in comparison with other viruses, and offer potential sites for antiviral intervention or development of gene therapy vectors. These herpesviruses are transported rapidly along microtubules (MTs) in the retrograde direction from the axon terminus to the dorsal root ganglion and then anterogradely in the opposite direction. Retrograde transport follows fusion and deenvelopment of the viral capsid at the axonal membrane followed by loss of most of the tegument proteins and then binding of the capsid via one or more viral proteins (VPs) to the retrograde molecular motor dynein. The HSV capsid protein pUL35 has been shown to bind to the dynein light chain Tctex1 but is likely to be accompanied by additional dynein binding of an inner tegument protein. The mechanism of anterograde transport is much more controversial with different processes being claimed for PrV and HSV: separate transport of HSV capsid/tegument and glycoproteins versus PrV transport as an enveloped virion. The controversy has not been resolved despite application, in several laboratories, of confocal microscopy (CFM), real-time fluorescence with viruses dual labelled on capsid and glycoprotein, electron microscopy in situ and immuno-electron microscopy. Different processes for each virus seem counterintuitive although they are the most divergent in the alphaherpesvirus subfamily. Current hypotheses suggest that unenveloped HSV capsids complete assembly in the axonal growth cones and varicosities, whereas with PrV unenveloped capsids are only found travelling in a retrograde direction.

HSV Encoded ICP-0 Inhibits TLR Signaling in CLL Cells by Targeting TRAF-6.

Innate immunity to viral vectors has largely been overlooked in development of gene therapy strategies and may play a key role in shaping immune responses and potentially clinical outcomes. In the setting of cancer immune therapy, the innate response may influence subsequent evolution of an anti-tumor adaptive immune response. We have studied two forms of HSV based amplicon vectors packaged in the presence or absence of HSV helper virus for use in immune therapy of chronic lymphocytic leukemia (CLL). Our previous work showed that the presence of helper-virus in HSV amplicon stocks impedes the development of tumor-specific adaptive immunity, and inhibits CTL development. We asked whether treatment outcomes could be explained by differences in the innate immune response elicited by the two vectors. We characterized innate immune response in CLL B cells transduced by helper-virus free (HF-HSV) vs. helper-virus containing (H+-HSV) amplicon vectors. Transduction of CLL B cells with HF-HSV induced robust innate cytokine/chemokine response that was not seen when transduction at similar efficiency was performed with H+-HSV vector. We profiled mRNA expression of TLR 1–10 in CLL B cells by quantitative PCR and confirmed the expression of TLR 2 and 9, which play a key role in mediating HSV innate response. Using 293 stably expressing TLR2/6 and TLR9, we show that the discrepancy in innate response of CLL B cells to transduction with HF-HSV and H+-HSV corresponded to differences seen in these viruses' capacity to activate TLR-2 and TLR-9. Pre-incubation with cyclohexamide restored H+-HSV capacity to activate TLR2 and 9 suggesting that H+-HSV encodes an inhibitory protein that interferes with TLR signaling. A screen of the four HSV immediate early (IE) proteins (ICP-0, ICP-22, ICP-27 and ICP-47) encoded by HSV Helper-virus identified ICP-0 as a likely candidate for inhibition of TLR signaling. Ectopic expression of ICP-0 inhibited innate response to HF-HSV in TLR2/6. In addition, ICP-0 inhibited other TLRs (TLR3, 4, 7, and 8) and IL-1 receptor mediated NF-κB activation, suggesting that ICP-0 targets a common signaling protein downstream of these receptors. Over-expression of various TLR signaling components identified TRAF-6 as a target for ICP-0 mediated inhibition of the NF-κB response. Finally, we correlated HSV vector-mediated innate response to capacity to prime an adaptive anti-CLL T cell response. A comparison of immune function of CD40L and B7.1 encoded by either hf-HSV or H+-HSV to prime autologous T cell response in vitro showed a clear advantage for HF-HSV amplicons encoding CD40L and B7.1 compared to H+-HSV amplicons. Our results highlight a novel mechanism through which HSV suppresses host innate immunity through a hitherto un-described ICP-0 inhibition of TLR/IL-1R signaling. The work also establishes a potential link between vector mediated innate immunity and ability to prime an adaptive anti-tumor immune response against CLL cells, which has important implications in relation to development of gene therapy vectors for clinical testing.

HSV trafficking and development of gene therapy vectors with applications in the nervous system

Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.

Electrochemical sensing of the behaviour of oligonucleotide lipoplexes at charged interfaces

Complexes between short oligodeoxynucleotides (ODN) with a variable dG x dC y base composition and liposomes composed of the cationic lipid DOTAP (ODN lipoplexes) were studied by differential pulse voltammetry at a glassy carbon electrode. Since lipoplexes are spontaneously formed by electrostatic interactions, the objective of the voltammetric study was to investigate their behaviour at the electrode surface/solution interface. It was verified that the peak current in the voltammograms for ODN lipoplexes was due to guanosine oxidation and that it was influenced both by the applied adsorption potential and the lipoplex (±) charge ratio used. It was found that for low ODN lipoplexes (±) charge ratios the peak current obtained was enhanced when compared to that registered with free ODN for the same concentration. This allowed a higher sensitivity in the determination of ODN by differential pulse voltammetry and a limit of detection of 5.5 ng/mL was achieved. A model that explains the organisation of ODN lipoplexes at the electrode surface/solution interface is proposed. The electrochemical results presented account for a better physicochemical characterisation of lipoplexes at charged interfaces, which can be important for the understanding and development of gene therapy vectors based on ODN lipoplexes.

Identification of Hyal2 as the cell-surface receptor for jaagsiekte sheep retrovirus and ovine nasal adenocarcinoma virus.

Jaagsiekte sheep retrovirus (JSRV) and ovine nasal adenocarcinoma virus (ONAV) replicate in the airway and cause epithelial cell tumors through the activity of their envelope (Env) proteins. Identification of the receptor(s) that mediate cell entry by these viruses is crucial to understanding the oncogenic activity of Env and for the development of gene therapy vectors based on these viruses that are capable of targeting airway cells. To identify the viral receptor(s) and to further study the biology of JSRV and ONAV, we developed retroviral vectors containing Moloney murine leukemia virus components and the Env proteins of JSRV or ONAV. We used a new technique involving positional cloning by phenotypic mapping in radiation hybrid cells to identify and clone the human receptor for JSRV, Hyal2, which also serves as the receptor for ONAV. Hyal2 is a glycosylphosphatidylinositol-anchored cell-surface protein that has low hyaluronidase activity and is a member of a large family that includes sperm hyaluronidase (Spam) and serum hyaluronidase (Hyal1). Hyal2 is located in a region of human chromosome 3p21.3 that is often deleted in lung cancer, suggesting that it may be a tumor suppressor. However, its role in JSRV or ONAV tumorigenesis, if any, is still unclear. JSRV vectors are capable of transducing various human cells, and are being further evaluated for gene therapy purposes.

179. Development of Gene Therapy Vectors Based on Chimpanzee Adenoviruses

179. Development of Gene Therapy Vectors Based on Chimpanzee Adenoviruses

Improvements in gene therapy: averting the immune response to adenoviral vectors.

Gene therapy is an interesting approach for the correction of defective genes, the treatment of cancer and the introduction of immunomodulatory genes. Various techniques for gene transfer into cells or tissues have been developed within the last decade; these can be divided generally into viral and nonviral gene transfer systems. Nonviral techniques include the liposome- or gene gun-mediated introduction of therapeutic genes; however, the efficiency of gene transfer by these applications is still very low. In contrast, viruses have optimised their strategies for efficient infection of virtually any cell type in a mammalian organism. The genetic modification of genomes from different virus families (Adenoviridae, Retroviridae, Herpesviridae) led to the development of gene therapy vectors with a similar capacity to infect cells or tissues as that of wild type viruses. In contrast to wild type viruses, gene therapy vectors are engineered to transfer therapeutic genes into the target cells or tissues. In addition, they have lost their capacity for replication in target cells, because of the removal of essential genes, which allows replication only in specialised packaging cell lines engineered for the production of recombinant viruses. Despite considerable progress over the past decade in the generation of gene transfer systems with reduced immunogenic properties, the remaining immunogenicity of many gene therapy vectors is still the major hurdle, preventing their frequent application in clinical trials. Recombinant adenoviruses have been shown to be promising vectors for gene therapy, since they are able to transduce both quiescent and proliferating cells very efficiently. However, a major disadvantage of adenoviral vectors lies in the activation of both the innate and adaptive parts of the recipient's immune system when applied in vivo. The inflammatory responses induced by adenovirus particles can be very strong and can be fatal in patients treated with these adenoviral constructs. Therefore, many experiments have been performed in the effort to prevent these inflammatory responses mediated by adenoviral particles. The depletion of cell populations responsible for these inflammatory responses as well as the application of immunosuppressive drugs have been investigated. Moreover, the generation of less immunogenic adenoviral vectors by further genetic modification within the adenoviral genome has led to vectors with reduced immunogenic properties. Both strategies to reduce inflammatory responses against adenoviral particles are discussed in this review.

FIV vector systems.

Why is feline immunodeficiency virus (FIV) such an appealing candidate for gene therapy vector development? Phylogenetic analysis suggests FIV is only distantly related to the primate lentiviruses, and despite repeated exposure, neither seroconversion nor other detectable evidence of human infection occurs. FIV naturally infects diverse Felidae worldwide, including the domestic cat. Here, the disease progression parallels the immunodeficiency caused by HIV, and for that reason, FIV and the cat provide an excellent model for anti-virals and AIDS vaccine research. Simple genome organization also facilitates vector development and analysis: FIV has only three accessory/regulatory proteins. To overcome FIV's cat-specific tropism, feline vectors are equipped with hybrid LTRs, since the FIV LTR shows low activity in human cells. Recombinant FIV vectors generate titers comparable to other lentiviral systems, are capable of incorporating heterologous envelopes and efficiently transduce dividing and nondividing cells in the presence and absence of the accessory proteins in vitro. Compared to HIV vectors, FIV vector development is still in its infancy, but initial in vivo data in various species and tissues indicate long-term gene expression at therapeutic levels, and thus FIV vectors hold great promise. Future efficacy studies in animal models and primates will determine the FIV vectors' suitability for gene therapy. The design of recombinant FIV vectors incorporates safety features described for primate lentiviral vectors with the benefit that biosafety testing of FIV vectors can occur in the natural host. Currently, FIV vectors are generated in a transient fashion, but the availability of a stable producer system amenable to better characterization and scale-up will considerably increase the potential for use of FIV vectors in the clinic.

TARGETING GENE THERAPY FOR TRANSPLANTATION WITH VENOM

TARGETING GENE THERAPY FOR TRANSPLANTATION WITH VENOM

A novel peptide, PLAEIDGIELTY, for the targeting of α 9β 1-integrins

Targeting gene therapy vectors to abundant receptors on airway epithelia may allow a significant enhancement of gene delivery and thereby be of particular importance for the gene therapy of cystic fibrosis. α 9β 1-Integrins are highly expressed throughout the human airway epithelia in vivo, irrespective of any particular clinical status. Aiming to improve the targeting of our non-viral integrin-mediated gene transfer systems to airway epithelia, we searched for a short tenascin C-derived peptide which would bind to these integrins. By utilizing recombinant bacteriophages that display overlapping regions of the third fibronectin type III repeat of tenascin C (TNfn3), we were able to localize its α 9β 1-integrin binding site to the B-C loop of TNfn3. A synthetic Pro-Leu-Ala-Glu-Ile-Asp-Gly-Ile-Glu-Leu-Thr-Tyr peptide (PLAEIDGIELTY) was shown to displace α 9β 1-integrin-expressing cells completely from binding to TNfn3. This peptide, therefore, may prove useful both for the examination of the functional importance of α 9β 1-integrins in vivo and the development of gene therapy vectors or drugs targeting these integrins.